METHOD FOR DETERMINING THE FREE ANTIGEN OF AN ANTIBODY IN A SAMPLE
20230393125 · 2023-12-07
Assignee
Inventors
Cpc classification
G01N33/6863
PHYSICS
International classification
Abstract
Herein is reported a method for determining free antigen of an antibody in an undiluted serum sample comprising the steps of a) applying the undiluted sample to a solid phase on which a capture antibody has been immobilized to form a capture antibody-antigen complex, wherein the capture antibody competes with the antibody for binding to a first epitope on the antigen, b) applying to the solid phase a tracer antibody to form a capture antibody-antigen-tracer antibody complex, wherein the tracer antibody specifically binds to a second epitope on the antigen, wherein the epitope of the tracer antibody is not overlapping with the epitope of the capture antibody on the antigen, and c) determining the free antigen of the antibody by determining the tracer antibody in the capture antibody-antigen-tracer antibody complex.
Claims
1. A method for determining free antigen, that can be specifically bound by an antibody, in an undiluted serum sample comprising free antigen, free antibody and antigen-antibody-complexes, wherein the method comprises the following steps: a) applying the undiluted serum sample to a solid phase on which a capture antibody has been immobilized to form a capture antibody-antigen complex, wherein the capture antibody competes with the antibody for binding to a first epitope on the antigen, b) applying to the solid phase a tracer antibody to form a capture antibody-antigen-tracer antibody complex, wherein the tracer antibody specifically binds to a second epitope on the antigen, wherein the epitope of the tracer antibody is not overlapping with the epitope of the capture antibody on the antigen, c) determining the free antigen of the antibody by determining the tracer antibody in the capture antibody-antigen-tracer antibody complex.
2. The method according to claim 1, wherein in step a) the applying is under conditions that at most 10% of the antibody bound to the antigen are replaced by the capture antibody, wherein in step a) at most 10% of the antibody bound to the antigen is replaced.
3. The method according to claim 1, wherein step a) is applying the undiluted serum sample to the solid phase on which a capture antibody has been immobilized to form a capture antibody-antigen complex, wherein the capture antibody competes with the antibody for binding to a first epitope on the antigen, wherein the sample is incubated with the solid phase for 240 seconds or less.
4. The method according to claim 1, wherein the half-life of the complex of the antigen binding site of the antibody specifically binding to the first epitope on the antigen and the antigen is 100 seconds or less.
5. The method according to claim 1, wherein the antibody is a bispecific antibody; wherein the bispecific antibody comprises a first antigen-binding site that specifically binds to the first epitope on the antigen and a second different antigen-binding site that specifically binds the second epitope on the antigen, wherein the tracer antibody competes with the bispecific antibody for binding to the second epitope on the antigen.
6. The method according to claim 5, wherein the half-life of the complex of the antigen binding site of the bispecific antibody specifically binding to the second epitope on the antigen and the antigen is 20 seconds or less.
7. The method according to claim 1, wherein the capture antibody and the tracer antibody is a non-human, non-humanized antibody.
8. The method according to claim 1, wherein the method is an enzyme-linked immunosorbent assay and the sample is incubated with the solid phase for 180 to 240 seconds.
9. The method according to claim 1, wherein the tracer antibody is incubated with the capture antibody-antigen complex for less than 1200 seconds.
10. The method according to claim 1, wherein the method is a nanoliter-scale, microfluidic, affinity flow-through format with laser-induced fluorescence detection and the sample is incubated with the solid phase for 2 seconds or less.
11. The method according to claim 1, wherein the tracer antibody is incubated with the capture antibody-antigen complex for less than 2 seconds.
12. The method according to claim 1, wherein the antibody is a therapeutic antibody.
13. The method according to claim 1, wherein the method is for determining the amount of free antigen, and step c) is determining the amount of free antigen of the antibody in the serum sample by determining the amount of the tracer antibody in the capture antibody-antigen-tracer antibody complex.
14. The method according to claim 1, wherein the antigen is human CCL2.
15. The method according to claim 1, wherein the antigen is human C5.
Description
DESCRIPTION OF THE FIGURES
[0438]
[0439]
[0440]
[0441]
[0442]
[0443]
[0444]
[0445]
[0446]
[0447]
[0448]
EXAMPLE 1
General Description of the Method According to the Invention
1) Nanoliter-Scale, Microfluidic, Affinity Flow-Through Format with Laser-Induced Fluorescence Detection Based Assay Format
[0449] To detect free antigen in human serum samples a Gyrolab® workstation assay was set-up. The test was used for quantitative detection of free antigen. Test samples, quality control samples and positive control standards were analyzed in 100% serum. Quality control samples and standards were prepared in 100% horse serum (comprising non-cross reactive endogenous target).
[0450] The following consecutive steps were performed:
[0451] Addition of capture reagent (mAb<target>rH-IgG-Bi, 5,000 ng/mL) followed by addition of sample and finally detection reagent (mAb<target>M-Alexa 647, 10,000 ng/mL). After each step a washing was performed to remove non-bound reagents. Each wash step consisted of the addition of a wash solution (1× PBS with 0.05% (v/v) Tween 20).
2) ELISA-Based Assay Format
[0452] To detect free antigen in human serum samples an ELISA-assay is set-up. The test is used for quantitative detection of free antigen. Test samples, quality control samples and positive control standards are analyzed in 100% serum. Quality control samples and standards are prepared in 100% horse serum (comprising non-cross reactive endogenous target).
[0453] The following consecutive steps are performed:
[0454] Different combinations of recombinant antigen and antibody are prepared in 100% horse pooled serum and incubated for 2 hours at RT. The calibration curve samples are prepared in 100% horse serum. Briefly, biotinylated (bispecific) therapeutic antibody as capture antibody, test sample and detection reagent (digoxygenylated (bispecific) therapeutic antibody are added stepwise to a 384-well streptavidin-coated microtiter plate and incubated on a non-vigorous shaker for 3-4 minutes (3.5 minutes as target). For detection of immobilized immune complexes, a polyclonal anti-digoxygenin-POD conjugate is added and the plate is incubated for 15-20 minutes. Optionally, the plate is washed three times after each step to remove unbound substances. ABTS is added to the plate and incubated at room temperature with shaking. Absorption is measured at 405/490 nm wavelength. The antigen concentrations are calculated based on the response of the calibration curve using the analytical software XLfit (IDBS).
EXAMPLE 2
Determination of KD Value in 100% Serum Sample
[0455] Based on the general method as described in Example 1 the following experiment was set up with the purpose to determine the KD value of a monoclonal antibody against its endogenous antigen. The calibration curve covered the range of 0.55 ng/mL to 3000 ng/mL. QC samples were prepared in horse serum at 5 concentrations. 0.55 ng/mL, 1.5 ng/mL, 100 ng/mL, 1200 ng/mL and 3000 ng/mL. Recovery of the QC samples met the criterion of +/−20% from the nominal value and were in the range of 97% to 112%. For determination of the KD value (following the method as outlined in WO 2014/023655) to endogenous target three human serum samples were diluted by a factor of 40 with horse serum to maintain 100% serum concentration and spiked with 1000 ng/mL, 2000 ng/mL, 3000 ng/mL, 4000 ng/mL and 5000 ng/mL of therapeutic antibody and equilibrated. A non-spiked human serum sample was analyzed with a dilution factor of 40 (with horse serum) as well to calculate the total target concentration in each individual serum sample. Based on the determined total target value, free target fraction was determined for each spiked serum sample. Based on each free fraction the KD value was calculated.
TABLE-US-00009 mAb<target> ID 1 ID 2 ID 3 concentration [ng/ml] KD [nM] KD [nM] KD [nM] 5000 0.049 0.076 0.048 4000 0.046 0.043 0.051 3000 0.056 0.041 0.042 2000 0.036 0.040 0.039 1000 0.046 0.072 0.060 average = 0.047 0.054 0.048 CV = 15% 34% 17%
[0456] The average (av) KD was 0.05 nM with a standard deviation (cv) of 0.012 nM.
EXAMPLE 3
Assay According to the Invention in 100% Human Serum
[0457] Based on Example 2 the following experiment was set up with the focus on the preparation and analysis of Free QC samples in 100% human serum. Free QC samples were prepared by addition of 100 μg/mL Crovalimab to each in Example 2 used individual human serum. Each serum sample was analyzed undiluted and free target concentration were analyzed as following: ID1=6.5 ng/mL; ID2=8.2 ng/mL; and ID3=7.6 ng/mL. Based on the KD value and total target concentration determined in Example 2 the estimated free target concentration should be in the range for ID1 of 2.41 ng/mL to 6.83 ng/mL, for ID2 in the range of 3.38 ng/mL to 9.56 ng/mL and for ID3 in the range of 3.51 ng/mL to 9.93 ng/mL. Calculation of the range is based on 2-fold standard deviation of the KD value. All samples of the three individual serum sample were within the calculated range, thus, verifying the correctness of the method according to the invention.
EXAMPLE 4
Comparative Example: ELISA in 25% Serum
[0458] Different combinations of recombinant cynomolgus CCL2 and biparatopic anti-CCL2 antibody CKLO2-SG1 were prepared in 100% horse pooled serum and incubated for 2 hours at RT. Samples were diluted 1 to 4 (MRD, resulting in 25% matrix) in assay buffer (PBS, 0.1% Tween, 1% BSA) and additionally 1 to 10 diluted in assay buffer containing 25% horse serum. The calibration curve was prepared in assay buffer containing 25% horse serum and covered the range of 7.8 to 1000 pg/mL CCL2 serum concentration. Briefly, biotinylated anti-CCL2 capture antibody (CNTO0888, CCL2-0004), test sample and detection reagent (digoxygenylated anti-CCL2 antibody (Humanized 11K2, CCL2-0002)), were added stepwise to a 384-well streptavidin-coated microtiter plate and incubated on a non-vigorous shaker for 1 hour, 12 to 14 minutes and 17 minutes respectively. For detection of immobilized immune complexes, a polyclonal anti-digoxygenin-POD conjugate was added and the plate was incubated for 20 minutes. The plate was washed three times after each step to remove unbound substances. ABTS was added to the plate and incubated at room temperature with shaking. Absorption was measured at 405/490 nm wavelength (see
TABLE-US-00010 ng/ml Ratio of determined CCL2 value dilution 1 to 40/ cyCCL2 Measured CCL2 value dilution 1 to 4 500.00 10.9 8.2 — — — — — — — 100.00 12.5 8.8 6.6 — — — — — — 20.00 — 8.4 6.2 5.2 — — — — — 4.00 — — 6.3 5.0 3.0 — — — — 0.80 — — — — 3.1 1.8 1.3 1.1 1.1 0.16 — — — — — — — — — 0.032 — — — — — — — — — 0 — — — — — — — — — 50000 12500 3125 781 195 49 12 3 0 ng/mL CKLO2-SG1
EXAMPLE 5
Assay According to the Invention in 100% Serum
[0459] A cynomolgus CCL2 calibration curve was prepared in 100% horse serum in a range of from 4 pg/mL to 1000 pg/mL CCL2 and analyzed in an ELISA format based on that as described in Example 4 but with the samples in 100% serum (no dilution). The incubation time of the sample on the assay plate was varied between 75 seconds and 12 minutes.
TABLE-US-00011 depicted mean AU values [n = 2] incubation time pg/ml 12 9 6 3 Min 1 Min cyCCL2 Min Min Min 30 s 15 s 1000.00 3.01 2.86 2.65 2.31 1.81 500.00 2.37 2.01 1.48 1.25 1.02 250.00 1.15 1.03 0.89 0.72 0.52 125.00 0.72 0.61 0.54 0.44 0.29 62.50 0.38 0.32 0.28 0.22 0.16 31.25 0.21 0.19 0.17 0.16 0.10 15.63 0.14 0.12 0.10 0.10 0.07 7.81 0.10 0.08 0.08 0.07 0.07 3.91 0.07 0.07 0.06 0.05 0.06 0 0.05 0.05 0.05 0.05 0.05
EXAMPLE 6
Method According to the Invention Using a Nanoliter-Scale, Microfluidic, Affinity Flow-Through Format with Laser-Induced Fluorescence Detection (Gyros Assay)
[0460] A Gyrolab™ based method was used in this example. Recombinant cynomolgus CCL2 samples were prepared in assay buffer (PBS, 0.1% (v/v) Tween-20, 1% BSA) and analyzed on a Gyrolab Xplore. The monospecific biotinylated parental anti-CCL2 antibody (CNT00888, alias CCL2-004) was used as capture reagent diluted to 1 μg/mL in assay buffer. For detection 1 μg/ml of the monospecific dig labeled anti-CCL2 antibody (Humanized 11K2, CCL2-0002) was pre-incubated with 1 μg/mL mAb<Dig>M-1.71.256-IgG-Alexa 647 for two hours in assay buffer. All reagents and samples were transferred to a 96-well PCR plate and loaded into the instrument together with a Gyrolab BioAffy 200 nL disc (Gyros Protein Technologies AB). A three-step assay protocol (200-3W-001) was selected. Briefly, the protocol describes the sequential addition of capture reagent, sample and detection reagent to designated streptavidin columns of the Gyrolab BioAffy 200 disc. Each reagent reaches the column at the same time after a short spinning step is applied to the disc. The columns were washed with PBS with 0.05 Tween after each step and finally laser induced fluorescence values were recorded within the instrument. A non-linear 4-parameter curve-fitting function (Wiemer-Rodbard) was applied to the averaged raw data to obtain a calibration curve. See also
TABLE-US-00012 cyCCL2c Fluorescence read out standard- coefficient signal to [pg/mL] replicate 1 replicate 2 average deviation of variance noise 40000 217.8 213.9 215.8 2.8 1.3% 450.2 20000 126.6 115.2 120.9 8.0 6.9% 252.2 10000 64.3 63.6 64.0 0.5 0.8% 133.4 5000 37.9 36.7 37.3 0.8 2.3% 77.8 2500 22.2 20.9 21.6 1.0 4.7% 45.0 1250 11.3 10.9 11.1 0.3 2.3% 23.2 625 6.0 6.1 6.0 0.0 0.5% 12.6 312.5 3.5 3.3 3.4 0.1 3.7% 7.2 0 0.5 0.5 0.5 0.0 2.1% 1.0
[0461] The assay was found to be linear over the selected assay range (312.5 pg/mL to 40,000 pg/mL).
EXAMPLE 7
Method According to the Invention Using a Nanoliter-Scale, Microfluidic, Affinity Flow-Through Format with Laser-Induced Fluorescence Detection (Gyros Assay) with Indirect and Direct Alexa Labelling of the Detection-Antibody According to the Invention
[0462] Humanized anti CCL2 antibody 11K2 (CCL2-0002) as well as rabbit anti CCL2 antibody 1H11 (CCL2-0011) were labeled with Alexa 647 (Molecular Probes, Invitrogen, Cat A20186). A recombinant cynomolgus CCL2 calibration curve in assay buffer (PBS, 0.1% Tween, 1% BSA) was analyzed with different anti CCL2 capture and detection reagents (1 μg/mL) as described for Example 6. [0463] Anti-CCL2 antibody-Bi (biotin-labeled anti-CCL2 antibody CNT00888), pre-incubated with humanized antibody 11K2 conjugated to digoxygenin and anti-Dig antibody M-1.71.256 (IgG) conjugated to Alexa 647 [0464] Anti-CCL2 antibody-Bi (CNT00888-Bi), pre-incubated humanized antibody 11K2 conjugated to Alexa 647 [0465] Rabbit anti-CCL2 antibody 2F6 conjugated to biotin (Bi; CCL2-0014), rabbit anti-CCL2 antibody 1H11 conjugated to Alexa 647
TABLE-US-00013 capture antibody: CNTO0888-Bi CNTO0888-Bi detection antibody: pre-incubated humanized 11K2-Dig + humanized mAb<Dig>M-1.71.256-IgG- cyCCL2 11K2-Alexa 647 Alexa 647 c [pg/mL] average cv average cv 51200 480.73 4.3% 266.68 0.8% 12800 200.33 0.5% 86.47 3.3% 3200 62.07 1.1% 27.75 2.9% 800 18.02 3.4% 7.86 6.9% 200 4.92 6.5% 2.38 0.7% 50 1.27 1.6% 0.88 4.3% 12.5 0.34 5.2% 0.50 3.9% 0 0.09 27.0% 0.39 6.3%
[0466] Compared to the pre-incubated detection reagents, sensitivity was increased when directly Alexa 647 labeled detection antibody was used (see Table above and
[0467] To avoid either false positive results that might occur due to bridging of ADAs directed against constant regions in capture and detection IgGs containing a human backbone as well as false negative results due to neutralizing ADAs directed against the CDRs of the therapeutic molecule that might cross react with the capture and detection reagents, competitive rabbit monoclonal antibodies were used. CCL2 values were back-calculated on the calibration curve and CCL2 recovery (% free) was calculated relative to the non-spiked 5 ng/mL CCL2 value (see also
TABLE-US-00014 capture antibody: CNTO0888-Bi 2F6-Bi detection antibody: cyCCL2 humanized 11K2-Alexa 647 1H11-Alexa 647 c [pg/mL] average cv average cv 7290 147.30 4.6% 151.05 1.8% 2430 57.60 4.1% 58.58 1.2% 810 20.75 2.6% 20.08 0.2% 270 7.10 3.4% 6.88 2.9% 90 2.54 0.6% 2.42 1.8% 30 0.87 1.6% 0.80 4.4% 10 0.34 1.3% 0.32 1.4% 0 0.06 36.3% 0.04 27.7%
[0468] The sensitivity to detect cyCCL2 was comparable between the described human parental capture and detection reagents and the selected competitive rabbit monoclonal anti CCL2 antibodies.
[0469] In the POC study four molecules were tested 1: CNTO0888-SG1 (=IgG1 wild type) anti-CCL2 antibody (n=3 animals) as control of maximal total CCL2 accumulation; group 2: a biparatopic anti-CCL2 antibody CKLO2-SG1 (IgG1 wild type) with pH dependent target binding but no Fc-modifications (n=3); group 3: a biparatopic anti-CCL2 antibody CKLO2-SG1100 with pH dependent target binding and Fc-pI and further modifications (n=4) and group 4: biparatopic anti-CCL2 antibody CKLO2-SG1095 with pH dependent target binding, Fc-pI and FcγRII and further modifications (n=4). These four molecules were pre-incubated in assay buffer with 5 ng/mL cyCCL2 for 2 hours in different concentrations and subsequently analyzed in the gyros assay including a cyCCL2 calibration curve. CCL2 values of samples were back-calculated on the calibration curve and recovery values (amount of free CCL2) were calculated relatively to 5 ng/ml CCL2.
TABLE-US-00015 CCL2 recovery; free [%] pg/mL ng/ml CNTO0888- CKLO2- CKLO2- CKLO2- CCL2 drug SG1 SG1 SG1100 SG1095 capture antibody: CNTO0888-Bi; detection antibody: 11K2-Alexa 647 5000 500 1.2% 0.4% 0.3% 0.3% 5000 250 2.1% 0.7% 0.5% 0.6% 5000 125 4.4% 2.5% 1.9% 2.0% 5000 62.5 13.3% 36.1% 28.8% 34.3% 5000 0 96.7% 104.9% 99.4% 106.3% capture antibody: 2F6-Bi; detection antibody: 1H11-Alexa 647 5000 500 1.1% 0.3% 0.3% 0.3% 5000 250 2.1% 0.7% 0.5% 0.6% 5000 125 4.7% 2.5% 2.0% 1.9% 5000 62.5 12.7% 36.1% 27.9% 33.3% 5000 0 92.7% 97.9% 97.7% 100.9%
[0470] Data depicted in the Table above show comparable results for the set-up with the human parental capture and detection molecules and the competitive rabbit mAbs.
EXAMPLE 8
Determination of Free CCL2 in POC Study of CCL2 Sweeping Efficiency in Cynomolgus Monkeys
[0471] Free CCL2 serum samples were analyzed with a non-validated, but qualified, Gyrolab™ immunoassay run on a Gyrolab Xplore. A biotinylated anti-CCL2 antibody (M-2F6-IgG) was used as capture reagent and for detection an Alexa 647 labeled anti-CCL2 antibody (M-1H11-IgG) was selected. Both reagents were diluted to 1 μg/mL in PBS, 0.1% Tween, 1% BSA and transferred to a 96-well PCR plate (Fisher Scientific). Cynomolgus monkey CCL2 calibration curve samples, QCs and undiluted serum samples were also transferred to a 96-well PCR plate. Both plates were loaded into the instrument together with a Gyrolab BioAffy 200 nL disc (Gyros Protein Technologies AB). A three-step assay protocol (200-3W-001) was selected. Briefly, the protocol describes the sequential addition of capture reagent, sample and detection reagent to designated streptavidin columns of the Gyrolab BioAffy 200 disc. Each reagent reaches the column at the same time after a short spinning step is applied to the disc. The columns were washed with PBS 0.05% Tween after each step and finally laser induced fluorescence values were recorded within the instrument. The free cynomolgus monkey CCL2 concentration was calculated based on the response of the calibration curve using XL Fit software (IDBS).
[0472] To demonstrate assay performance QC samples (High QC 1820 pg/mL cyCCL2, Mid QC 230 pg/mL cyCCL2 and LQC 30 pg/mL CCL2) were prepared in 1× PBS, 0.1% Tween, 1% BSA and analyzed in each run in parallel to cynomolgus pool serum (obtained from biotrend). Calibrators were also prepared in 1× PBS, 0.1% Tween, 1% BSA in the range of 2430 pg/mL to 10 pg/mL. Additionally cynomolgus pooled serum (CPS) was spiked with 7.5 ng/mL and 10 μg/mL CNTO0888 as well as 15 ng/mL and 10 μg/mL CKLO2-SG1095. These samples were also analyzed as QC samples in the respective assay runs (dependent on the group). As shown in the following Table free CCL2 value variation between the assay runs was below 11% for these free QC samples. The assay QCs containing recombinant CCL2 in assay buffer were found to be within +/−20% of the nominal concentration in all 12 assay runs (data not shown).
TABLE-US-00016 Measured CCL2 [pg/mL] CPS with CKLO2-SG1095 CPS with CNTO0888 Run CPS 10 μg/mL 15 ng/ml 10 μg/mL 7.5 ng/ml 1 586.7 13.5 128.8 — — 2 597.3 13.3 134.1 — — 3 560.5 12.0 144.0 — — 4 570.5 14.8 116.7 — — 5 600.5 13.4 130.6 — — 6 586.2 — — 15.1 272.6 7 593.3 — — 13.7 264.0 8 563.0 — — 14.8 242.7 9 657.4 14.4 139.4 — — 10 568.6 17.1 142.0 — — 11 573.0 12.2 122.1 — — 12 552.5 14.6 135.1 — — av 584 14 133 15 260 cv 5% 11% 7% 5% 6% —: not determined
EXAMPLE 9
Mouse Study
[0473] To support studies conducted in B16-huCCL2/CCL2-null mouse models the assay described in Example 8 was performed with recombinant human wild-type CCL2 as calibrator (see
TABLE-US-00017 Fluor Read Hu wtCCL2 out Gyros c [pg/mL] average (n = 3) cv in % 21870 233.45 2.4% 7290 98.02 8.3% 2430 37.35 7.5% 810 13.46 9.5% 270 4.72 1.0% 90 1.65 5.5% 30 0.61 12.3% 10 0.23 4.5% 0 0.06 2.6%
[0474] As controls for study pooled mouse serum (MPS) was either spiked with 5 ng/mL recombinant human wild-type CCL2 or with 5 ng/mL recombinant human wild-type CCL2 and 5 μg/mL or 50 ng/mL CKLO2-SG1095. Recovery values were calculated relative to the nominal 5 ng/ml. The corresponding data is shown in the Table below.
TABLE-US-00018 Measured CCL2 Recovery [pg/mL] (%) MPS_5 ng/mL huCCL2 5139 102.8% MPS_5 ng/mLCCL_5 μg/mLCKLO2- Below lower limit of SG1095 quantification (10 pg/mL) MPS_5 ng/mLCCL_50 ng/mLCKLO2- 1668 33.4% SG1095
EXAMPLE 10
Method According to the Invention for the Determination of Free C5 in 100% Serum Samples
[0475] A Gyrolab® workstation was used. 20 μL of test samples, quality control samples, blank samples and each positive control standard were transferred into the designated wells of a multi-well plate. Thereto the respective capture and detection reagent was added. The sealed plate was centrifuged for 10 sec. at at least 3000 g. A 3-step method with two wash solutions for needle washes was used for the analysis in the Gyrolab® workstation. Samples, blanks, quality controls and standards were measured in replicates (N=2).
[0476] Result interpretation of samples is based on qualitative interpretation of the FU of the samples and corresponding quality controls (QCs). 1% PMT Fluorescence raw data were exported as Excel File using Gyrolab® Evaluator Software. A standard calibration curve was generated by a non-linear 4-parameter fit using a Wiemer-Rodbard function (e.g. using XLfit for MS Excel): Wiemer Rodbard: [y(x)={(1*A)+((B−A)/{1+{(C/x)AD))))]. A and B are responsible for signal deviation (approximated start and end of the calibration curve). C and D are responsible for the curve shape. Quantitation of the results relative to the positive control antibody is done by back-calculation of the mean signals of the samples using the fitted calibration curve. Representative raw date (fluorescent unit) of the calibration are shown in the following Table.
TABLE-US-00019 Serum concentration [ng/mL] 3000 714 170 40 9.6 2.3 0.55 blank Average 387 129 30 7.2 1.7 0.38 0.08 0.02 emission [FU]