Immunoassay for the determination of Fc-region modified antibodies

Abstract

Herein is reported a method for the determination of the amount of a bivalent antibody in a serum or plasma sample obtained from a non-human experimental animal, whereby the antibody comprises one or more mutations in the Fc-region compared to the corresponding wild-type Fc-region that has a sequence of SEQ ID NO: 01, 02, or 03, wherein the method comprises the following steps a) immobilizing a non-antibody polypeptide to which more than one copy of the antigen of the antibody is covalently conjugated on a solid surface, b) incubating the immobilized antigen with the sample to form an immobilized antigen-antibody complex, c) incubating the immobilized antigen-antibody complex with the antigen conjugated to a detectable label to form an immobilized ternary complex, and d) determining the amount of the antibody by determining the amount of the detectable label in the immobilized ternary complex.

Claims

1. A method for the determination of the amount of an anti-hapten non-therapeutic Fc-modified bivalent antibody in a serum or plasma sample obtained from a non-human experimental animal, whereby the antibody comprises one or more mutations in the Fc-region compared to the corresponding wild-type Fc-region that has a sequence of SEQ ID NO: 01, 02, or 03, wherein detection of the antibody is independent from of Fc region modification, wherein the method comprises the following steps in the following order: (a) providing a biotinylated bovine plasma albumin (RPLA) to which more than one copy of the hapten antigen is chemically conjugated thereon to form a biotinylated bovine plasma albumin (RPLA)-hapten capture reagent, (b) immobilizing the capture reagent of (a) on a streptavidin or avidin conjugated on a solid surface, wherein the biotinylated bovine plasma albumin (RPLA)-hapten capture reagent has a coating density of about 50 ng/ml, (c) incubating the capture reagent of (b), with the sample comprising the anti-hapten non-therapeutic Fc-modified bivalent antibody to form an immobilized antigen-antibody complex, wherein the anti-hapten antibody is selected from the group consisting of an anti-biotin antibody, an anti-digoxygenin antibody, an anti-theophylline antibody, an anti-helicar antibody, and an anti-bromodeoxyuridine antibody, (d) incubating the immobilized antigen-antibody complex of (c) with the hapten antigen of said antibody, wherein the hapten is conjugated to a detectable label to form an immobilized ternary complex, (e) determining the amount of the antibody by determining the amount of the detectable label in the immobilized ternary complex; and (f) evaluating the antibody in the experimental animal for pharmacokinetic properties associated with Fc-region mutations, wherein the antibody is quantified at serial time points by performing steps (a)-(e), wherein the hapten antigen is not present in the experimental animal from which the analyzed sample is obtained.

2. The method according to claim 1, wherein the detectable label is an enzyme.

3. The method according to claim 1, wherein the detectable label is a peroxidase.

4. The method according to claim 3, wherein the detectable label is horseradish peroxidase.

5. The method according to claim 2, wherein the concentration of the detectable label is about 300 mU/mL.

6. The method according to claim 3, wherein the determining is by incubating the immobilized ternary complex with 3,3′,5,5′-tetramethyl benzidine.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 Scheme of a standard immunoassay.

(2) FIG. 2 Scheme of the immunoassay as reported herein.

(3) FIG. 3 Assay standard curve.

(4) FIG. 4 Pharmacokinetic analysis of a wild-type antibody (diamond) and LALA-PG-AAA mutant (square).

(5) FIG. 5 Standard curves.

(6) The following examples, figures and sequences are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

(7) Materials

(8) TABLE-US-00001 Device Type Manufacturer fluorescence spectrometer Infinite F200 PRO Tecan Group AG SPR BIAcore T100 GE Healthcare photometer Sunrise Tecan Austria GmbH pipettes 1-, 8-, 12-channel pipettes Eppendorf AG shaker Thermo-Shaker PHMP-4 Grant-bio washer hydroFlex Tecan Austria GmbH centrifuge Galaxy Mini VWR International Type Manufacturer BIAcore T100 control GE Healthcare i-control 1.10 Tecan Group AG Microsoft Office 2010 Microsoft WinNonline 5.3 Pharsight XLfit4 IDBS ChemSketch 12.0 Advanced Chemistry Development Inc. 10× PBS Roche Diagnostics GmbH ABTS Solution Roche Diagnostics GmbH Beagle-poolserum Bioreclamation LLC. Bi(XOSu)-RPLA-Dig(XOSu) Roche Diagnostics GmbH BM Blue POD Substrate (TMB) Roche Diagnostics GmbH Bronidox (=5-Bromo-5-nitro-1,3- Sigma Aldrich dioxane) Cynomolgus-poolserum Sera Laboratories International Ltd Dig-3-CME-UEEK-Bi Roche Diagnostics GmbH Dig(XOSu)-POD Roche Diagnostics GmbH HPPA Sigma-Aldrich Human-poolserum (n = 20; 10 Trina Bioreactives AG female, 10 male) mAk<Dig>H-IgG LALA Roche Diagnostics GmbH mAk<Dig>H-IgG LALA PG Roche Diagnostics GmbH mAk<Dig>H-IgG LALA PG AAA Roche Diagnostics GmbH mAk<Dig>H-IgG wt Roche Diagnostics GmbH mAk<Dig>M-19.11.-IgG Roche Diagnostics GmbH mouse-poolserum Roche Diagnostics GmbH Minipig-Individual plasma F. Hoffmann-La Roche AG Minipig-poolplasma Roche Diagnostics GmbH pAk<Dig>S-Fab-POD Roche Diagnostics GmbH pAk<M-Fc>Rb-IgG-HRP Jackson Immuno Research Laboratories INC. Polystyrol-MTP Maxisorb Thermo Fisher Scientific Inc./NUNC RPLA-Dig(XOSu) Roche Diagnostics GmbH Streptavidin-coated MTP MicroCoat Streptavidin-coated MTP C96 white Greiner Bio-One GmbH Streptavidin-POD conjugate Roche Diagnostics GmbH Tween 20 Calbiochem non-coated pre-incubation-MTP Thermo Fisher Scientific Inc./NUNC (polypropylene) ELISA universal buffer Roche Diagnostics GmbH hydrogen peroxide 30% Merck KgaA Type Composition Assay-buffer Universal buffer for ELISA Coating-buffer 0.1M NaHCO.sub.3, pH 9.6 Blocking-buffer 1× PBS, 5% RPLA1, Bronidox 0.002% stopping buffer 0.5M H.sub.2SO.sub.4 TRIS-buffer 12.14 g Tris + 3.5 ml HCL (ad 1l), pH 8.6 Universal buffer — Wash buffer 1× PBS, 0.05% Tween 20, 0.002% Bronidox

Example 1

Immunoassay

(9) The method as reported herein is a non-competitive enzyme linked immunosorbent assay in sandwich format. This assay setup allows for the detection of non-biologically active antibodies in 10% matrix (serum).

(10) A streptavidin-coated 96-well plate (SA-MTP) was coated with activated biotin (biotin-(XOSu)-RPLA-digoxigenin(XOSu) (BI-RPLA-DIG). The coating was done with 100 μL of a solution (50 ng/mL BI-RPLA-DIG) added to each well for one hour at room temperature with shaking (500 rpm). Thereafter the wells are washed three times with 300 μL wash buffer each.

(11) For analysis standard samples, quality control samples and samples were analyzed in duplicates. Standard samples and quality control samples were prepared in 100% matrix and diluted afterwards 1:10 (v/v) with assay buffer. The standard samples were prepared using the same material also applied to the experimental animal in a 1:1 dilution series in 100% matrix as follows: The first standard sample (STD A) was prepared using a monoclonal murine anti-digoxigenin antibody (mAb<Dig>M-19.11-IgG) at a concentration of 350 ng/mL in 100% matrix. By diluting STD A with 100% matrix STD B was obtained and so on up to STD G (350 ng/mL—5.5 ng/mL). The eighth standard is non-spiked 100% matrix. The standard samples and the blank were diluted 1:10 (v/v) with assay buffer. Each sample and standard was added in a volume of 100 μL to the wells. The plate was incubated for one hour at room temperature with shaking (500 rpm). Thereafter the supernatant was removed and the wells were washed three times with 300 μL per well wash buffer. To each well 100 μL of the detection solution (digoxygenin(XOSu)-peroxidase (DIG_POD); 300 mU/mL) was added. Thereafter the plate was incubated for one hour at room temperature with shaking (500 rpm). After removal of the supernatant each well was washed three times with 300 μL was buffer each. The color reaction was initiated by addition of 100 μL of a 3,3′,5,5′-tetramethyl benzidine (TMB) solution. After 18 minutes the reaction was stopped by the addition of 0.5 mol/L phosphoric acid and extinction determined at 450 nm with a reference value taken at 690 nm.

Example 2

(12) Capture Reagent

(13) 1) Non-Specific Adsorption

(14) The capture reagent digoxygenin-conjugated bovine plasma albumin (RPLA-DIG) was used in this example.

(15) A solution comprising 1.5 μg/mL RPLA-DIG in 100 mM sodium hydrogen carbonate buffer (pH 9.6) was prepared. Each well of a polystyrol multi-well plate was filled with 200 μL of this solution and incubated for one hour with shaking. Thereafter the supernatant was removed and the wells were washed three times each with 300 μL/well blocking buffer comprising bovine serum albumin. After removal of the supernatant the wells are washed three times each.

(16) Standard samples were prepared using a 1:1 dilution series in 100% assay buffer as follows: The first standard sample (STD A) was prepared using a monoclonal murine anti-digoxigenin antibody (mAb<Dig>M-19.11-IgG) at a concentration of 100 ng/mL in assay buffer. By diluting STD A with assay buffer 1:1 (100% dilution) STD B was obtained and so on up to STD G. The eighth standard is non-spiked assay buffer.

(17) Six standard series in duplicate were determined (incubation time in the well one hour).

(18) For detection a solution comprising a horseradish peroxidase conjugated rabbit polyclonal anti-Fc-antibody (pAk<M-Fc>Rb-IgG-HRP) was prepared (50 mU/mL). This detection solution is diluted to 25 mU/mL, 12.5 mU/mL, 6.25 mU/ml, 3.13 mU/mL and 1.56 mU/mL so that six detection solutions with different detection reagent concentrations were obtained.

(19) After removing the supernatant from the wells and three-time washing the detection reagent is added to the wells. Therefore for each of the six standard series 100 μL of the respective dilution of the detection reagent is added. After the incubation the supernatant is removed and an ABTS solution is added. The produced colored reaction product is determined at 405 nm with a reference wavelength of 490 nm.

(20) TABLE-US-00002 POD-conc. [mU/ml] 50 25 12.5 6.25 3.13 1.56 t [min] 4.83 8.97 17.25 35.18 57.95 57.95 STD Ext. CV Ext. CV Ext. CV Ext. CV Ext. CV Ext. CV [ng/ml] [OD] [%] [OD] [%] [OD] [%] [OD] [%] [OD] [%] [OD] [%] 100 2.406 1 2.419 0 2.451 2 2.386 3 1.896 2 1.037 2 50.0 2.085 1 2.035 1 2.025 1 2.033 1 1.609 1 0.867 0 25.0 1.768 2 1.637 0 1.597 1 1.599 1 1.218 0 0.650 0 12.5 1.247 2 1.093 0 1.058 1 1.070 1 0.789 3 0.424 2 6.25 0.780 2 0.647 0 0.621 2 0.636 1 0.466 4 0.259 6 3.13 0.437 0 0.362 2 0.346 5 0.350 4 0.267 2 0.149 0 1.56 0.232 1 0.195 1 0.186 3 0.197 1 0.152 2 0.093 3 0 0.021 3 0.022 3 0.025 3 0.032 0 0.037 0 0.038 2 S:N 102 95 83 64 43 23
2) Specific Adsorption

(21) Two different capture reagents for specific and directed adsorption were prepared: i) with defined BI-DIG stoichiometry: DIG-3-CME-UEEK-Bi (DIG=digoxygenin; 3-CME=3-carboxymethyl ether; UEEK=β-alanine-glutamic acid-glutamic acid-lysine; BI=biotin; MW approx. 1.1 kDa); denoted as BI-DIG in the following) ii) with undefined BI-DIG stoichiometry: BI-RPLA-DIG (RPLA=bovine plasma albumin; MW approx. 70 kDa).

(22) As standard sample an anti-ANG2 antibody has been used in this example.

(23) The capture reagents were each incubated over night with the standard and first detection reagent (see previous example) incubated. The incubation is performed in polypropylene plates in which no non-specific adsorption to the surface occurs.

(24) For detection a digoxigenylated anti-idiotypic antibody is added to the overnight pre-incubated sample (mAb<ID-mAb<ANG2>>M.2.6.81-IgG(SPA)-Dig(XOSu) (DIG-IGG)). The signal was developed using a peroxidase conjugate polyclonal anti-digoxygenin antibody (pAk<DIG>S-Fab-POD).

(25) The capture reagent BI-DIG was used at a concentration of 219.99 ng/mL and the capture reagent BI-RPLA-DIG was used at a concentration of 14 μg/mL. With this concentrations the capture reagents were present during the pre-incubation in a 100-fold molar excess (BI-100) compared to the other reagents. In a 1:10 (BI-10) and 1:100 dilution (BI-1) solutions with a 1:10 and 1:1 molar ratio were obtained. Analogously the first detection reagent was used at a concentration of 30 μg/mL (DIG-100) resulting in a molar ratio of capture reagent to detection reagent of 1:100. In a 1:10 (DIG-10) and 1:100 dilution (DIG-1) solutions with a 1:10 and 1:1 molar ratio were obtained. The analyte was employed at a concentration of 3 μg/mL (STD-10). A 1:10 dilution provided STD-1.

(26) The pre-generated immune complex containing solution was applied to the wells of a streptavidin-coated multi-well plate and the secondary detection reagent against the Fc-region of the analyte (pAb<M-Fc>Rb-IGG-HRP; HRP=horseradish peroxidase) was added in a concentration of 160 ng/mL. After an incubation time of one hour the substrate solution was added and the generated colored reaction product determined photometrically.

(27) The results for the BI-DIG conjugate determined at 18.5 min are shown in the following table.

(28) TABLE-US-00003 DIG-0 DIG-10 STD- STD- STD- STD- STD- STD- 0 1 10 0 1 10 BI-0 0.025 0.026 0.026 0.060 0.043 0.027 BI-1 0.023 0.025 0.024 0.046 0.039 0.036 BI-10 0.023 0.027 0.029 0.043 0.036 0.376 BI-100 0.024 0.028 0.035 0.042 0.035 0.399 BI-0 0.024 0.025 0.025 0.223 0.221 0.112 BI-1 0.025 0.025 0.029 0.217 0.213 0.122 BI-10 0.025 0.036 0.144 0.236 0.218 0.140 BI-100 0.028 0.032 0.246 0.257 0.226 0.172 DIG-1 DIG-100

(29) The results for the BI-RPLA-DIG conjugate determined at 18.5 min are shown in the following table.

(30) TABLE-US-00004 DIG-0 DIG-10 STD- STD- STD- STD- STD- STD- 0 1 10 0 1 10 BI-0 0.030 0.029 0.030 0.070 0.060 0.036 BI-1 0.027 0.033 0.032 0.051 0.057 1.565 BI-10 0.027 0.032 0.041 0.054 0.063 1.754 BI-100 0.028 0.032 0.037 0.056 0.057 0.086 BI-0 0.030 0.029 0.032 0.254 0.287 0.213 BI-1 0.029 0.118 0.424 0.261 0.261 0.225 BI-10 0.029 0.041 0.962 0.301 0.332 0.362 BI-100 0.033 0.035 0.042 0.469 0.465 0.523 DIG-1 DIG-100

(31) It can be seen that only for high concentrations the digoxigenylated antibody binds non-specifically to the plate. It can further be seen that the extinction maximum for both capture reagents is in the same molar ratio. In case of the BI-DIG is the molar ratio of the capture reagent is shifted by a factor of 10 up compared to the BI-RPLA-DIG. The direct comparison of the signal-to-noise ratio (S/N) of the extinction maximum shows that for the Bi-RPLA-Dig the S/N is 32.8 (S/N=1.754/0.054), whereas for the BI-DIG is 9.5 (S/N=0.399/0.042).

Example 3

(32) Assay-Setup

(33) Different variants of assay setups are compared in this example: i) serial setup (application of capture reagent, analyte and detection reagent, each step is separated by one washing step), ii) pre-incubation of analyte and detection reagent, and iii) pre-incubation of capture reagent, analyte and detection reagent.

(34) 1) Serial Setup

(35) The results obtained with the serial setup after 18.6 min are shown in the following table. The maximum of 36.3 has been obtained with an analyte concentration of 50 ng/mL.

(36) TABLE-US-00005 c(Bi- RPLA- c(Dig-POD) [mU/mL] Dig) 25 50 75 100 150 300 [ng/mL] S:N (at 50 ng/ml analyte) 400 5.1 5.9 6.0 6.5 6.7 8.1 200 9.9 11.4 11.9 12.9 13.8 16.4 100 17.7 19.4 19.7 21.3 22.4 25.1 75 22.6 25.5 25.6 27.2 26.7 32.4 50 25.8 30.2 28.7 30.5 31.0 36.3 25 25.7 29.0 28.4 29.5 32.2 35.3 10 16.3 20.2 18.8 18.6 19.9 20.4 5 8.8 10.3 9.6 9.4 11.4 11.3

(37) It can be seen that with decreasing BI-RPLA-DIG concentration and increasing DIG-POD concentration the S/N increases. At a concentration of the detection reagent of 300 mU/mL and of the capture reagent of 50 ng/mL a maximum of 36.3 can be seen.

(38) In the following Table (determined for a detection reagent concentration of 300 mU/mL) it can be seen that the extinction increases up to a capture concentration of 50 ng/mL and decreases thereafter. Above this concentration avidity effects occur resulting in a decreased signal due to bivalent binding.

(39) TABLE-US-00006 c(Dig-POD) [mU/ml] 0 0.5 5.0 50 500 5000 STD Ext. CV Ext. CV Ext. CV Ext. CV Ext. CV Ext. CV [ng/ml] [OD] [%] [OD] [%] [OD] [%] [OD] [%] [OD] [%] [OD] [%] 50.0 0.047 20 0.113 0 0.679 3 2.087 0 0.412 10 0.061 2 25.0 0.036 6 0.089 4 0.440 3 1.073 2 0.208 4 0.045 2 12.5 0.036 4 0.076 7 0.287 0 0.544 0 0.120 2 0.040 4 6.25 0.035 4 0.055 3 0.176 1 0.283 1 0.075 2 0.037 2 3.13 0.035 6 0.046 3 0.109 0 0.161 3 0.056 0 0.034 0 1.56 0.036 2 0.043 5 0.072 0 0.097 1 0.046 2 0.034 4 0.78 0.030 14 0.037 4 0.054 1 0.066 1 0.038 2 0.029 2 0 0.035 10 0.037 2 0.038 0 0.039 2 0.036 2 0.033 0

(40) It can be seen from the data in the following Table (detection after 18.5 min incubation time) that there is no difference in the dynamic range, i.e. no limitation due to the DIG-POD component. When assuming that above a signal twice the blank signal a good quantification is possible it can be seen that the sensitivity is independent of the DIG-POD concentration. Thus, the serial format using ABTS as color reagent has with a DIG-POD concentration of 300 mU/mL and 50 ng/mL capture reagent a detection range of from 1.56 ng/mL to 200 ng/mL.

(41) TABLE-US-00007 c(mAk DIG-POD [mU/mL] <DIG> 300 600 900 M-IgG) Ext. CV Ext. CV Ext. CV [ng/mL] [OD] [%] [OD] [%] [OD] [%] 6400 2.897 3 2.950 1 2.874 0 3200 2.805 4 2.875 0 2.796 2 1600 2.806 2 2.852 1 2.831 2 800 2.673 1 2.741 0 2.766 2 400 2.486 0 2.537 2 2.603 0 200 2.277 2 2.342 0 2.436 1 100 1.721 0 1.769 0 1.958 0 50.0 1.107 0 1.127 1 1.328 2 25.0 0.595 1 0.621 1 0.751 1 12.5 0.304 1 0.316 0 0.394 4 6.25 0.168 0 0.172 0 0.214 0 3.13 0.099 1 0.105 1 0.128 1 1.56 0.063 2 0.069 2 0.079 2 0.78 0.046 0 0.051 1 0.055 1 0.39 0.036 4 0.040 4 0.055 31 0 0.028 8 0.032 9 0.036 10

(42) The reaction has been repeated using TMB as color substrate. It has been found that when using TMB instead of ABTS the assay can be performed in a shorter time, i.e. it is quicker, and higher extinction values can be obtained. The detection range using TMB is 0.78 ng/mL to 50 ng/mL. The values are shown in the following Table.

(43) TABLE-US-00008 c(mAk DIG-POD [mU/mL] <DIG> 300 100 50 M-IgG) Ext. CV Ext. CV Ext. CV [ng/mL] [OD] [%] [OD] [%] [OD] [%] 6400 3.499 6 3.686 0 3.569 5 3200 3.465 9 3.138 0 3.426 0 1600 3.508 11 3.734 0 3.551 4 800 3.506 5 3.301 0 3.521 4 400 3.603 13 3.435 0 3.349 4 200 3.490 5 3.464 5 3.358 2 100 3.296 5 3.258 0 3.103 3 50.0 2.596 1 2.239 1 2.046 1 25.0 1.405 1 1.176 0 1.076 2 12.5 0.693 1 0.573 0 0.530 4 6.25 0.348 1 0.292 0 0.268 3 3.13 0.184 1 0.157 0 0.149 1 1.56 0.107 0 0.095 1 0.085 2 0.78 0.068 3 0.062 0 0.056 1 0.39 0.050 6 0.046 0 0.044 6 0 0.033 2 0.032 0 0.030 0
2) Pre-Incubation Analyte and Detection Reagent

(44) From the following Table can be seen that at a concentration of 50 ng/mL of the monoclonal anti-digoxygenin antibody the S/N increases with increasing coating concentration and concomitant reduced detection reagent. At 400 ng/mL BI-RPLA-DIG and 31.3 mU/mL DIG-POD a maximum is reached. The color reagent was ABTS.

(45) TABLE-US-00009 c(BI- RPLA- c(DIG-POD) [mU/mL] DIG) 1000 500 250 125 62.5 31.3 [ng/mL] S/N 400 3.4 5.4 11.1 19.6 38.0 53.3 200 2.5 5.0 9.3 18.5 34.9 51.0 100 2.0 3.1 5.6 10.7 23.8 42.2 75 1.3 2.9 5.1 10.2 21.3 38.9 50 1.6 2.2 4.4 7.2 16.9 33.1 25 1.3 1.8 2.6 4.2 9.4 20.0 10 1.1 1.2 1.7 2.5 4.5 10.1 5 1.1 0.9 1.3 1.7 2.8 6.3

(46) In all three DIG-POD concentrations tested in the following series a hook-effect starting at the anti-digoxygenin antibody concentration of 400 ng/mL can be seen. It can further be seen that with increasing DIG-POD concentration the saturation plateau is getting broader and the hook effect is reached later. A clear limitation by the POD concentration can be seen. For this assay at a BI-RPLA-DIG concentration of 400 ng/mL and a DIG-POD concentration of 30 mU/mL the assay has a detection range of from 3.13 ng/mL to 150 ng/mL. The other two concentrations are not suitable due to the limited POD effect and the longer required time.

(47) TABLE-US-00010 c(mAk DIG-POD [mU/mL] <DIG> 30 15 7.5 M-IgG) Ext. CV Ext. CV Ext. CV [ng/mL] [OD] [%] [OD] [%] [OD] [%] 6400 1.352 2 0.739 1 0.401 1 3200 1.880 2 1.134 0 0.640 0 1600 2.405 1 1.676 1 1.048 0 800 2.751 0 2.212 2 1.553 0 400 2.704 0 2.249 1 1.683 2 200 2.563 1 2.047 1 1.486 2 100 1.898 1 1.668 2 1.232 3 50.0 0.919 1 1.170 0 0.969 2 25.0 0.392 1 0.647 12 0.692 3 12.5 0.160 2 0.227 1 0.287 3 6.25 0.077 2 0.098 1 0.102 3 3.13 0.044 2 0.050 1 0.053 3 1.56 0.031 2 0.032 0 0.033 4 0.78 0.026 0 0.027 3 0.025 6 0.39 0.023 0 0.023 0 0.023 3 0 0.022 0 0.023 3 0.022 6
3) Pre-Incubation of Capture Reagent, Analyte and Detection Reagent

(48) From the data in the following Table it can be seen that at a concentration of 50 ng/mL of the monoclonal anti-digoxygenin antibody the S/N increases with decreasing capture reagent concentration as well as decreasing detection reagent concentration. At a concentration of 13.7 ng/mL of BI-RPLA-DIG and 18.8 mU/mL of DIG-POD a maximum is reached. The color reagent was ABTS.

(49) TABLE-US-00011 c(BI- RPLA- c(DIG-POD) [mU/mL] DIG) 600 300 150 75 37.5 18.8 [ng/mL] S/N 10000 6.1 3.6 2.5 1.8 1.5 1.1 3333 22.0 14.0 9.5 5.5 3.9 2.5 1111 30.9 26.4 25.6 16.4 11.4 6.3 370 34.0 39.9 36.1 28.8 21.5 13.0 123 20.7 28.3 36.9 39.4 35.6 25.3 41.2 9.3 15.0 25.2 36.1 47.3 47.5 13.7 4.1 6.6 10.1 22.1 37.2 48.8 4.57 1.1 1.2 1.0 1.0 1.0 0.8

(50) From the following data set it can be seen that at a BI-RPLA-DIG concentration of 10 ng/mL to a DIG-POD concentration of 16.6 mU/mL a limitation occurs and the standard curve does not reach an OD value of 2. For this combination also a hook effect is visible at an analyte concentration of 200 ng/mL. As the data is comparable the criterion is the reagent use for selecting one of these in this category. No hook effect is detectable up to an analyte concentration of 6400 ng/mL. The detection range at a BI-RPLA-DIG concentration of 1000 ng/mL and a DIG-POD concentration of 600 mU/mL is 3.13 ng/mL to 200 ng/mL. The same detection range has an assay using 100 ng/mL BI-RPLA-DIG and 100 mU/mL DIG-POD.

(51) TABLE-US-00012 DIG-POD [mU/mL] 600 100 16.6 c(mAk BI-RPLA-DIG [ng/mL] <DIG> 1000 100 10 M-IgG) Ext. CV Ext. CV Ext. CV [ng/mL] [OD] [%] [OD] [%] [OD] [%] 6400 3.005 0 2.933 0 0.294 0 3200 2.973 0 2.940 1 0.415 0 1600 3.054 1 3.023 1 0.573 1 800 2.931 0 2.975 1 0.739 1 400 2.788 1 2.915 0 0.874 1 200 2.243 2 2.420 1 1.018 1 100 1.350 3 1.339 2 1.065 1 50.0 0.804 0 0.650 3 0.781 2 25.0 0.404 2 0.346 2 0.331 1 12.5 0.207 1 0.154 2 0.130 1 6.25 0.118 3 0.086 2 0.064 3 3.13 0.073 4 0.053 3 0.040 4 1.56 0.049 9 0.037 4 0.029 10 0.78 0.038 4 0.030 0 0.025 9 0.39 0.031 0 0.025 0 0.021 17 0 0.026 3 0.024 0 0.023 9

Example 4

(52) Different Color Reagents

(53) The extinction value shaded in the next Table for the different color reagents ABTS and TMB and the emission values for HPPA, respectively, are below the detection limit and cannot be quantified. It can be seen that the assay in the serial setup (the assay as reported herein) has a detection limit using TMB of 0.94 ng/mL in is thereby two times more sensitive compared to ABTS. Using HPPA the detection limit is 0.47 ng/mL (using the standard series with 5 U/mL DIG-POD). Although this assay variant is more sensitive than the assay using TMB surprisingly the assay signal could not be obtained in a sufficiently reproducible manner to allow the setup of a robust assay.

(54) TABLE-US-00013 ABTS: c(Dig-POD) [mU/ml] 15 10 5 1 0.5 0.1 STD Ext Ext Ext Ext Ext Ext. [ng/ml] [OD] S:N [OD] S:N [OD] S:N [OD] S:N [OD] S:N [OD] S:N 60.0 2.207 13.3 2.074 16.8 1.905 20.9 1.530 47.1 1.370 39.1 1.025 40.2 30.0 1.362 8.2 1.220 9.9 1.066 11.7 0.794 24.4 0.682 19.5 0.485 19.0 15.0 0.858 5.2 0.723 5.9 0.585 6.4 0.405 12.5 0.346 9.9 0.238 9.3 7.50 0.503 3.0 0.380 3.1 0.318 3.5 0.213 6.5 0.184 5.3 0.131 5.1 3.75 0.352 2.1 0.260 2.1 0.204 2.2 0.122 3.8 0.104 3.0 0.076 3.0 1.88 0.281 1.7 0.190 1.5 0.138 1.5 0.078 2.4 0.066 1.9 0.049 1.9 0.94 0.200 1.2 0.176 1.4 0.111 1.2 0.055 1.7 0.052 1.5 0.037 1.5 0 0.166 1.0 0.124 1.0 0.091 1.0 0.033 1.0 0.035 1.0 0.026 1.0

(55) TABLE-US-00014 TMB: c(Dig-POD)[mU/ml] 15 10 5 1 0.5 0.1 STD Ext Ext Ext Ext Ext Ext. [ng/ml] [OD] S:N [OD] S:N [OD] S:N [OD] S:N [OD] S:N [OD] S:N 60.0 3.585 4.0 3.506 6.7 3.516 13.5 3.476 44.6 3.395 58.5 3.146 71.5 30.0 3.447 3.9 3.364 6.4 3.229 12.4 2.888 37.0 2.608 45.0 1.951 44.3 15.0 2.990 3.4 2.755 5.2 2.354 9.0 1.669 21.4 1.421 24.5 0.982 22.3 7.50 2.282 2.6 1.870 3.6 1.359 5.2 0.881 11.3 0.748 12.9 0.510 11.6 3.75 1.656 1.9 1.223 2.3 0.905 3.5 0.489 6.3 0.398 6.9 0.271 6.2 1.88 1.190 1.3 0.957 1.8 0.631 2.4 0.293 3.8 0.236 4.1 0.156 3.5 0.94 0.808 0.9 0.803 1.5 0.390 1.5 0.202 2.6 0.147 2.5 0.099 2.3 0 0.856 1.0 0.526 1.0 0.261 1.0 0.078 1.0 0.058 1.0 0.044 1.0

(56) TABLE-US-00015 HPPA: c(Dig-POD) [mU/ml] STD 10 5 1 0.5 0.1 0.05 [ng/ml] [FU] S:N [FU] S:N [FU] S:N [FU] S:N [FU] S:N [FU] S:N 15.0 44824 26.7 38558 52.6 27790 83.0 23087 95.0 14700 61.2 11761 50.5 7.50 24711 14.7 19836 27.0 13058 39.0 10388 42.7 6497 27.1 4927 21.1 3.75 13976 8.3 10101 13.8 6227 18.6 4843 19.9 2839 11.8 2144 9.2 1.88 7604 4.5 5106 7.0 2765 8.3 2145 8.8 1158 4.8 882 3.8 0.94 4726 2.8 3077 4.2 1267 3.8 950 3.9 516 2.1 428 1.8 0.47 2558 1.5 1558 2.1 571 1.7 426 1.8 293 1.2 266 1.1 0.23 1677 1.0 1009 1.4 381 1.1 315 1.3 257 1.1 238 1.0 0 1681 1.0 734 1.0 335 1.0 243 1.0 240 1.0 233 1.0

Example 5

(57) Matrix Range

(58) It can be seen from the following Table that in different matrices the spiked standard samples compared to the standard samples produced with buffer only the obtained signal is reduced. Referencing to the buffer samples the spiked standards are recovered with 94% to 72% from the different sample matrices. The variation is 3% to 5%. Thus, there is no significant difference between all the matrices.

(59) TABLE-US-00016 c(mAk <DIG> buffer human minipig cynomolgus mouse beagle M-IgG) Ext. recov. Ext. recov. Ext. recov. Ext. recov. Ext. recov. Ext. recov. [ng/mL] [OD] [%] [OD] [%] [OD] [%] [OD] [%] [OD] [%] [OD] [%] STA D: 35.0 1.627 100 1.392 86 1.374 85 1.255 77 1.414 87 1.311 81 STD B: 17.5 0.810 100 0.681 84 0.691 85 0.662 81 0.708 87 0.661 81 STD C: 8.75 0.419 100 0.361 85 0.365 86 0.347 81 0.368 87 0.342 80 STD D: 4.38 0.229 100 0.199 84 0.208 89 0.194 82 0.206 88 0.190 79 STD E: 2.39 0.134 98 0.115 77 0.125 89 0.117 80 0.123 86 0.112 74 STD F: 1.09 0.090 101 0.078 74 0.086 93 0.080 79 0.083 86 0.077 72 STD G: 0.55 0.067 101 0.056 55 0.066 94 0.061 77 0.061 77 0.061 77 blank 0.044 — 0.041 — 0.047 — 0.044 — 0.046 — 0.045 —

(60) TABLE-US-00017 matrix average c(mAk<DIG>M- Ext. recov. SD CV IgG) [ng/mL] [OD] [%] [OD] [%] STA D: 35.0 1.349 83 0.065 5 STD B: 17.5 0.680 84 0.020 3 STD C: 8.75 0.357 84 0.011 3 STD D: 4.38 0.199 84 0.008 4 STD E: 2.39 0.118 81 0.006 5 STD F: 1.09 0.081 81 0.004 5 STD G: 0.55 0.061 76 0.003 5 blank 0.044 — 0.002 5

Example 6

(61) Assay Characteristics

(62) In FIG. 3 standard curve for the assay as reported herein is presented comprising STD A to STD G and a blank value. The detection range is from 0.55 ng/mL to 35 ng/mL based on the weight of the employed anti-digoxygenin antibody. The following Table provides the respective data.

(63) TABLE-US-00018 assay conc. 35 17.5 8.75 4.38 2.19 1.09 0.55 0 [ng/mL] plasma 350 175 87.5 43.8 21.9 10.9 5.5 0 conc. [ng/mL] average 1.903 1.036 0.539 0.288 0.164 0.103 0.071 0.042 signal [OD] SD signal 0.030 0.044 0.027 0.015 0.007 0.006 0.003 0.004 [OD]

(64) The assay as reported herein has an intra-assay precision with a standard deviation/coefficient of variation of 2% to 6%. The intra-assay recovery rate is between 100% and 104%.

(65) The assay as reported herein has an inter-assay precision with a standard deviation/coefficient of variation of 5% to 12%. The inter-assay recovery rate is between 78 and 117%. The calculated recover rate for the μL- and LLQC samples is between 87% and 121% and, thus, within the acceptable range of 25%.

Example 7

(66) Selectivity

(67) From the following Table can be seen that the assay has a very good selectivity. The extinction values determined for the different antibodies are well below the lowest standard sample approximately at blank sample level. Thus, capture and tracer reagent are not linked to each other up to a concentration of 10 μg/mL. Thus, the assay is specific for the anti-digoxygenin antibody.

(68) TABLE-US-00019 analyte STD-conc. extinction [OD] conc. [ng/mL] STD A-H A1 A3 A5 A7 [ng/mL] 35.0 1.873 0.048 0.052 0.046 0.045 10000 17.5 1.044 0.045 0.044 0.038 0.050 1000 8.75 0.541 0.054 0.046 0.041 0.040 100 4.38 0.293 0.042 0.042 0.040 0.041 10 2.19 0.160 0.044 0.051 0.041 0.041 10000 1.09 0.097 0.044 0.043 0.042 0.042 1000 0.55 0.071 0.044 0.043 0.042 0.049 100 0 0.044 0.052 0.046 0.043 0.048 10 A2 A4 A6 A8

(69) The assay as reported herein shows no hook effect up to a concentration of 100 μg/mL antibody in 100% pooled plasma (assay concentration 10 μg/mL). The following Table provides the respective determined values. Samples (if applicable after dilution) are detected within the acceptance interval of 80% to 120% recovery (AQL=above limit of quantification; BQL=below limit of quantification).

(70) TABLE-US-00020 plasma dilution factor to highest conc. signal calc. conc. recovery concentrated sample [ng/mL] [OD] [ng/ml] [%] — 100000 3.504 ALQ — 10 10000 3.658 ALQ — 100 1000 3.464 ALQ — 300 333.3 2.032 384.3 115.3 900 111.1 0.728 126.8 114.1 2700 37.04 0.274 43.30 116.9 8100 12.35 0.113 14.26 115.5 24300 4.115 0.061 BLQ — recovery range [%] — — 114.1-116.9

(71) The dilution linearity of the assay as reported herein has been tested in plasma concentrations of up to 100 μg/mL anti-digoxygenin antibody in eight minipig individual plasmas. 41 out of the 45 diluted samples have been detected within the acceptance interval of 80% to 120% (all 45 dilutions in the interval of 81% to 138%). The different plasma matrices have no significant influence on the recovery of the analyte.

Example 8

(72) Sample Quantification

(73) From the following Tables it can be seen that by exchanging individual amino acid residues in the Fc-region of antibodies the pharmacokinetic half-life has been changed (see FIG. 4). The data has been generated by determining in 16 different minipig sera. Shaded values are below the limit of quantification, ivt denotes intravitreal application and iv denotes intravenous application.

(74) TABLE-US-00021 sampling c(mAk<Dig>H-IgG1 wt) [ng/mL] time point group 4 (ivt) group 7 (iv) [h] 19 20 21 22 23 24 29 30 0.5 — — — — — — 7931 7808 6 — — — — — — 7246 7383 12 132 174 559 109 112 14.7 5351 5673 24 235 416 758 194 341 57.6 4197 4641 48 377 621 953 382 602 151 — — 72 463 667 923 427 560 258 3031 3564 168 710 892 958 606 828 768 2220 — 336 — — 932 25.8 273 1089 1722 — 504 — — — — −4.6 1431 574 — 672 — — — — −1.9 1225 70.9 —

(75) TABLE-US-00022 sampling c(mAk<Dig>H-IgG1_LALA_PG_AAA) [ng/mL] time point group 3 (ivt) group 6 (iv) [h] 13 14 151 61 17 18 27 28 0.5 — — — — — 7,691 7,142 6 — — — — — — 4,984 4,891 12 269 21.2 99.1 300 58.5 91.3 3,279 3,104 24 304 102 179 612 95.5 162 2082 1989 48 319 141 268 654 196 239 — — 72 259 143 208 567 230 232 560 514 168 79.0 373 155 349 316 288 111 125 336 — — 110 243 170 161 15.4 0.0 504 — — — — 103 31.0 6.3 0.0 672 — — — — 70.4 16.9 4.5 0.0

(76) The three Fc-region modified antibodies have been detected using the assay as reported herein. The standard curve is shown in FIG. 5 and the respective date in the Table below.

(77) TABLE-US-00023 LALA- LALAPG- LALAPGAAA- mutant mutant mutant wild- calc. calc. calc. type conc. recovery conc. recovery conc. Recovery ng/mL ng/ml % ng/ml % ng/ml % 250 193 77 251 100 230 92 125 95.6 76 131 105 122 98 62.5 47.8 77 67.1 107 61.5 98 31.3 25.2 81 32.2 103 33.6 107 15.6 12.5 80 16.6 106 16.7 107 7.81 6.67 85 8.38 107 8.38 107 3.91 4.02 103 4.14 106 4.14 106

LITERATURE

(78) Carl Roth GmbH und Co. KG: Albumine für die Biochemie und Molekularbiologie. ILK January 2009 Diamandis E. P., Christopoulos T. K. (1991) The biotin-(strept)avidin system: principles and applications in biotechnology. Clinical Chemistry, 37, 625-636 European Medicines Agency (July 2011), Guideline on bioanalytical method validation Goebel-Stengel M, Stengel A, Taché Y, Reeve J R (2011) The importance of using the optimal plastic ware and glassware in studies involving peptides. Analytical Biochemistry, 414, 38-46 Hoffmann-La Roche AG: Roche Lexikon—Medizin. 5. Ed. Munich: Urban & Fischer, 2003 Holländer, Georg: Immunologie—Grundlage für Klinik und Praxis. 1. Ed. Munich: Urban & Fischer, 2006 Issaq H J, Xiao Z, Veenstra T D (2007) Serum and Plasma Proteomics. Chemical Reviews, 107(8), 3601-3620 Luttmann et al.: Der Experimentator—Immunologie. 3. Ed. Heidelberg: Spektrum Akademischer Verlag, 2009 Mould D. R., Green B. (2010) Pharmacokinetics and pharmacodynamics of monoclonal antibodies: concepts and lessons for drug development. BioDrugs, 24(1), 23-39 Raem, A.; Rauch, P.: Immunoassays. 1. Ed. Munich: Spektrum Akademischer Verlag, 2007 Simpson J. R., Greening D. W: Serum/Plasma Proteomics. 1 vol. Totowa: Humana Press Inc., 2011 Stubhan, Miriam: Das Göttinger Minipig als Telemetriemodell für pharmakologische Zwecke, Diss. Univ. Munich, 2008 U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Veterinary Medicine (May 2001), Guidance for Industry—Bioanalytical Method Validation

ABBREVIATIONS

(79) ABTS 2,2′-Azino-di-(3-ethylbenzthiazoline-6-sμLfonic acid) Ak antibody ALQ Above limit of quantification AP Assay buffer (=Universal-buffer) AS amino acid Bi Biotin BLQ Below limit of quantification BSA Bovine serum albumin CDR Complementarity determining region CV Coefficient of variation Dig Digoxigenin ELISA Enzyme Linked Immunosorbent Assay F(ab′).sub.2 Fragment antigen binding (bivalent) Fab Fragment antigen binding (monovalent) FC Fragment Crystallizable H Human HPPA 3-(4-Hydroxyphenyl)-propionic acid HRP Horseradish peroxidase Ig Immunoglobulin IgA Immunoglobulin of subclass A IgD Immunoglobulin of subclass D IgE Immunoglobulin of subclass E IgG Immunoglobulin of subclass G IgM Immunoglobulin of subclass M M mouse mAk monoclonal antibody MTP micro titer plate OD optical density pAk polyclonal antibody POD Peroxidase PP Polypropylene PS Polystyrol Rb Rabbit RFU Relative fluorescence units RPLA bovine plasma albumin SA Streptavidin S:N Signal-to-noise ratio SD Standard deviation STD Standard TMB 3,3′,5,5′-Tetramethylbenzidine XOSu X(ε-Aminocaproic acid)-O-Succinimide