A novel sandwich ELISA for the quantitative and sensitive determination of IL-13 in human serum and plasma was established. The assay employs an incubation step at acidic pH, which was shown to decrease nonspecific binding and interference from IL-13 binding proteins. The assay was validated and was shown to be accurate and precise over the entire quantification range (0.59 to 68.4 pg/mL in human EDTA plasma). The validated assay was successfully applied to samples from healthy volunteers and patients with atopic seasonal rhinitis. The assay is suitable for use in clinical trials to monitor efficacy or pharmacodynamic effects of drug candidates.
Interleukin-13 (IL-13) is an immunoregulatory cytokine secreted by T helper 2 lymphocytes. As being involved in the activation of IgE production, eosinophil infiltration, mucus hyper secretion, and subepithelial fibrosis, IL-13 is a central mediator in the inflammation of airways and in the pathogenesis of asthma and allergy [
In human, IL-13 has already been measured in bronchoalveolar lavage fluid (BAL) allowing to distinguish asthmatic children from control subjects [
Here we report the development and validation of a sensitive, accurate, and reproducible assay for the quantification of IL-13 levels in human plasma and serum. The assay employs the sandwich ELISA technique built on commercially available reagents. Samples are incubated with the capture antibody at acidic pH to strip IL-13 from any of its binding partners. We applied the validated assay to measure total circulating IL-13 in atopic patients and compared to levels obtained in apparently healthy controls.
Immobilizer Amino 96-well microtiter plates were purchased from Nunc (Roskilde, Denmark). Antibodies and IL-13 standard protein were taken from the human IL-13 Module Set (Bender Medsystems, Vienna, Austria). IL-13R
Human serum and plasma samples used in assay development were obtained from the Etablissement français du Sang d’Alsace (Strasbourg, France) and from an internal blood donation program (Novartis AG, Basel, Switzerland). They were collected on presumably healthy and untreated individuals.
Human plasma samples for assay validation and sample analysis were obtained from Bioreclamation Inc., both from healthy and atopic subjects. A donor is determined to be “healthy” based on criteria set by either the Food and Drug Administration or the American Association of Blood Banks (AABB).
Human plasma samples from atopic subjects were collected based on following criteria set: male or female subjects aged between 18 and 60 years (inclusive) and in good health as determined by past medical history, physical examination, vital signs, electrocardiogram, and laboratory tests. Subjects must have a history of atopy, defined as a history of seasonal allergic rhinitis for at least 2 years (in relation to the ragweed season), and evidence of atopy, defined as a positive skin prick test (wheal difference allergen—negative control ≥3 mm) to ragweed allergen at screening.
For serum collection, whole blood was drawn into a dry collection bag and allowed to clot overnight in a refrigerator and spun to serum at 2,800 ×g for 20 minutes in a refrigerated centrifuge (5°C).
For EDTA plasma collection, whole blood was drawn into a collection bag containing anticoagulant and mixed gently for 5 minutes. The blood was allowed to sit at room temperature for 30 minutes and spun to plasma at 2,800 ×g for 20 minutes in a refrigerated centrifuge (5°C).
All samples were obtained with the appropriate informed consents.
Calibrators samples (Cs) were prepared freshly everyday by successive dilution of IL-13 standard protein in substitute matrix. Substitute matrix was made of 80% FBS/20% human serum. Human serum used in substitute matrix was selected for having a nondetectable endogenous IL-13 level.
Cs were incubated for 1 h with gentle shaking at room temperature before dilution (see next paragraph).
All samples (Cs, quality controls (QCs) and unknown samples) are first diluted 1 : 2 in one of the following buffers to reach, respectively, pH 7.4, pH 5.0, or pH 3.5: PBS/0.1% Tween20/6% BSA (pH 7.4), MES 1 M/0.1% Tween20/6% BSA (pH 4.9), HCl/glycine 3 M/0.1% Tween20/6% BSA (pH 3.4).
QCs and unknown samples are then further diluted up to 1 : 3 or more in the following buffers with corresponding pH values: PBS/0.05% Tween20/3% BSA/50% FBS (pH 7.4), MES 0.5 M/0.05% Tween20/3% BSA/50% FBS (pH 5.0), HCl/glycine 1.5 M/0.05% Tween20/3% BSA/50% FBS (pH 3.5).
All samples are analyzed by ELISA immediately after dilution.
This protocol describes the ELISA procedure used during assay development. For assay validation, a few modifications were made to the procedure and are reported wherever applicable.
A mouse monoclonal anti-human IL-13 capture antibody was covalently coupled to microtiter plates. To do so, a 2.5
Nonspecific binding (NSB) signal was obtained after the incubation of a plasma or serum sample, diluted into the appropriate buffer solution, in a well devoid of capture antibody.
The validation of the IL-13 assay under acidic conditions was evaluated by assessing calibration curve, intrarun accuracy and precision, interrun accuracy and precision, sensitivity/working range, parallelism and selectivity of IL-13, interference with IL-13R alpha2, and stability in biological matrix. The approach for assay validation was detailed in a previous report [
The IL-13 concentration was determined in duplicate in human plasma samples by using IL-13 assay under acidic conditions (pH 3.5). Each sample analysis run contained a calibration curve, a zero standard, and three levels of QC samples covering the assay working range. Two independent preparations of each QC level were assayed in duplicate. The QCs and samples were diluted 1 : 2 according to the minimum required dilution (MRD). The sample measurement was considered acceptable only when acceptance criteria for both Cs and QCs are met and the CV% of the duplicate measurement was less than or equal to 30%, and the two values of the duplicate measurements were equal or above the LLOQ. Values measured below the LLOQ were assigned the value LLOQ/2 (0.3 pg/mL).
We evaluated binding activity of rhIL-13 to immobilized capture antibody at different incubation pH values: pH 7.4, pH 5, pH 3.5, and pH 3.25. The obtained titration curves were displayed in Figure
Background signal and detection limit of rhIL-13 obtained at different incubation pH values.
pH 7.4 | pH 5 | pH 3.5 | pH 3.25 | |
---|---|---|---|---|
Background signal (OD495) | 0.260 | 0.115 | 0.108 | 0.105 |
Detection limit (pg/mL) | 0.31 | 0.16 | 0.31 | 5.00 |
Titration curves of rhIL-13 incubated at pH 7.4, pH 5, pH 3.5, and pH 3.25 on immobilized capture antibody.
Background signal (OD) is the signal generated with all reagents but in absence of the specific analyte, the IL-13 protein. Detection limit is the lowest calibration point for which the concentration can be back-calculated on the regression curve with 80 to 120% accuracy and with CV below 20%.
Binding activity of rhIL-13 to capture antibody was not affected at pH 5 as compared to more classical and physiological conditions (pH 7.4). However, background signal was reduced by half. At pH 3.5, binding activity of rhIL-13 was strongly reduced: the signal generated by 40 pg/mL of rhIL-13 was twice as less as at pH 5. Nevertheless, detection limit remained low, at 0.31 pg/mL. At lower pH (pH 3.25), rhIL-13 could be detected only starting from 5 pg/mL, meaning that capture by the immobilized antibody at this level of concentration was not possible or negligible at this pH.
At pH 7.4 (classical incubation conditions for an ELISA), the NSB observed with plasma samples was high and variable from one sample to another (Figure
NSB obtained in plasma samples at pH 7.4, pH 5, and pH 3.5. NSB is the signal obtained by the detection (biotinylated anti-IL13 antibody and enzyme linked streptavidin) of plasma components nonspecifically bound to the plate surface in the absence of capture antibody.
The high NSB observed at pH 7.4 indicated that measures obtained at this pH in plasma samples could not be considered as specific to the presence of IL-13. Hence, in the following paragraphs, measures of IL-13 concentration obtained at pH 7.4 were not reported.
In the previous paragraphs, it has been shown that incubation of plasma samples at pH 5 or pH 3.5 allowed to reduce nonspecific signal while keeping good assay sensitivity. In this paragraph, endogenous concentrations of IL-13 were measured at these pH values on three independent runs in order to evaluate assay reproducibility (Figure
Interrun precision of endogenous IL-13 measurement in two human plasma samples at pH 5 and pH 3.5. The endogenous IL-13 concentration was measured at pH 5 and pH 3.5 in three independent runs. Precision (% CV) of the three measures was calculated for the two plasma samples.
The interference of recombinant soluble human IL-13 R
Recovery of rhIL-13 concentration in the presence of its receptor rhIL-13R
A human serum was spiked with 10 pg/mL rhIL-13 and high concentrations of rhIL-13R
At pH 5, the measure of 10 pg/mL rhIL-13 was inhibited in the presence of its receptor rhIL13R
At pH 3.5, recovery of rhIL-13 in the presence of rhIL-13R
The effect of rhIL-13R
Recovery of endogenous IL-13 concentration in the presence of rhIL-13R
These results showed that acidic conditions (pH 3.5) were able to modify interactions between IL-13, binding proteins, and assay antibodies, so that the total (bound and free) concentration of IL-13 was measured. The improvement of assay precision on endogenous IL-13 measurement at pH 3.5 may be linked to the reduction of binding proteins interference. Besides, sample acidification allowed to reduce nonspecific signal generated by matrix components as shown in Section
A calibration curve was run on each 96-well assay plate using the IL-13 standard provided with the assay kit. The curve was based on 12 calibrators (Cs) of recombinant human IL-13 diluted in 11 calibrators diluents (at pH 3.5) in the range of 0.16 to 40.00 pg/mL. Calibration curve data is shown in Table
Typical IL-13 calibration curve data obtained during assay validation.
IL-13 standard concentration (pg/mL) | Wells | OD | OD mean | CV (%) | BackCalc mean | BackCalc Cv in % | Accuracy (%) | Accuracy mean (%) |
---|---|---|---|---|---|---|---|---|
40 | A11 | 3.038 | 3.073 | 1.6 | 39.6 | 2.86 | 97.05 | 99.05 |
A12 | 3.108 | 101.06 | ||||||
30 | B11 | 2.543 | 2.592 | 2.6 | 30.2 | 3.9 | 97.78 | 100.56 |
B12 | 2.64 | 103.33 | ||||||
20 | C11 | 1.897 | 1.926 | 2.1 | 20.2 | 2.65 | 99.27 | 101.16 |
C12 | 1.955 | 103.06 | ||||||
10 | D11 | 1.024 | 1.058 | 4.5 | 10.1 | 5.04 | 97.07 | 100.66 |
D12 | 1.091 | 104.25 | ||||||
5 | E11 | 0.556 | 0.559 | 0.5 | 4.9 | 0.62 | 97.48 | 97.91 |
E12 | 0.561 | 98.34 | ||||||
2.5 | F11 | 0.333 | 0.328 | 1.8 | 2.5 | 2.46 | 102.45 | 100.7 |
F12 | 0.324 | 98.94 | ||||||
1.25 | G11 | 0.21 | 0.213 | 1.5 | 1.3 | 2.93 | 98.51 | 100.6 |
G12 | 0.215 | 102.68 | ||||||
0.63 | H11 | 0.161 | 0.159 | 1.6 | 0.6 | 4.81 | 104.68 | 101.23 |
H12 | 0.158 | 97.79 | ||||||
0.31 | D9 | 0.135 | 0.137 | 2.2 | 0.3 | 11.35 | 102.43 | 111.37 |
D10 | 0.139 | 120.31 | ||||||
0.21 | E9 | 0.124 | 0.125 | 0.7 | 0.2 | 6.75 | 83.33 | 87.51 |
E10 | 0.126 | 91.69 | ||||||
0.16 | F9 | 0.123 | 0.123 | 0.5 | 0.1 | 5.82 | 96.95 | 93.12 |
F10 | 0.122 | 89.29 |
Neat plasma samples were tested pure and after dilution (direct or serial) to assess the endogenous levels of IL-13 and to evaluate the parallelism of the assay. Six different lots of human plasma samples were freshly diluted in sample diluent (pH 3.5) at 1 : 2, 1 : 3, 1 : 4, and 1 : 6 dilutions. The accuracy of the IL-13 determination in each individual diluted sample was within the range of 82.0 to 124.8% as compared to the values determined at dilution 1 : 2 (Table
Parallelism assessment of IL-13 assay in human plasma under acidic conditions. The accuracy was calculated using the IL-13 concentration determined at dilution 1 : 2 as a reference value. Parallelism was considered acceptable when accuracy was between 70 and 130% for at least 5/6 samples at each dilution.
Dilution | Raw result (pg/mL) | Accuracy ( | |
---|---|---|---|
Observed ( | Expected ( | ||
Sample 1 | |||
1 : 2 | 0.425 | ||
1 : 3 | 0.250 | 0.283 | 88.2 |
1 : 4 | 0.195 | 0.213 | 91.8 |
1 : 6 | N.C | N.C. | N.C. |
Sample 2 | |||
1 : 2 | 1.280 | ||
1 : 3 | 0.760 | 0.853 | 89.1 |
1 : 4 | 0.525 | 0.640 | 82.0 |
1 : 6 | 0.362 | 0.427 | 84.8 |
Sample 3 | |||
1 : 2 | 0.745 | ||
1 : 3 | 0.517 | 0.497 | 104.0 |
1 : 4 | 0.425 | 0.373 | 114.1 |
1 : 6 | 0.285 | 0.248 | 114.8 |
Sample 4 | |||
1 : 2 | 1.190 | ||
1 : 3 | 0.763 | 0.793 | 96.2 |
1 : 4 | 0.540 | 0.595 | 90.8 |
1 : 6 | 0.365 | 0.397 | 92.0 |
Sample 5 | |||
1 : 2 | 1.330 | ||
1 : 3 | 1.020 | 0.887 | 115.0 |
1 : 4 | 0.830 | 0.665 | 124.8 |
1 : 6 | 0.553 | 0.443 | 124.8 |
Sample 6 | |||
1 : 2 | 0.270 | ||
1 : 3 | 0.207 | 0.180 | 114.8 |
1 : 4 | 0.148 | 0.135 | 109.3 |
1 : 6 | N.C | N.C. | N.C. |
N.C.: value not calculated as being outside the calibration range.
The same 6 different lots of human plasma were spiked with 25.0 pg/mL of recombinant human IL-13, and accuracy was assessed at 1 : 2, 1 : 3, 1 : 4, 1 : 12, and 1 : 36 with assay buffer. The spiking solution represented <10% of the final volume. The accuracy of measurement of spiked plasma samples was ranging from 97.8 to 137.2% as shown in Table
Selectivity assessment of IL-13 assay in human plasma under acidic conditions. 25.0 pg/mL of recombinant IL-13 was spiked in the same 6 plasma samples tested for parallelism. Accuracy was calculated using the IL-13 concentration determined at dilution 1 : 2 as a reference value. Selectivity was considered acceptable when accuracy was between 70 and 130% for at least 5/6 samples at each dilution.
Dilution | Raw result (pg/mL) | Accuracy ( | |
---|---|---|---|
Observed ( | Expected ( | ||
Sample 1 | |||
1 : 2 | 14.11 | ||
1 : 3 | 9.36 | 9.41 | 99.5 |
1 : 4 | 7.70 | 7.06 | 109.1 |
1 : 12 | 2.42 | 2.35 | 102.8 |
1 : 36 | 0.80 | 0.78 | 102.5 |
Sample 2 | |||
1 : 2 | 13.99 | ||
1 : 3 | 9.64 | 9.32 | 103.4 |
1 : 4 | 7.25 | 6.99 | 103.6 |
1 : 12 | 2.48 | 2.33 | 106.4 |
1 : 36 | 0.85 | 0.78 | 109.5 |
Sample 3 | |||
1 : 2 | 11.12 | ||
1 : 3 | 8.28 | 7.41 | 111.7 |
1 : 4 | 6.47 | 5.56 | 116.4 |
1 : 12 | 2.45 | 1.85 | 132.2* |
1 : 36 | 0.85 | 0.62 | 137.2* |
Sample 4 | |||
1 : 2 | 15.17 | ||
1 : 3 | 9.89 | 10.11 | 97.8 |
1 : 4 | 7.46 | 7.59 | 98.4 |
1 : 12 | 2.54 | 2.53 | 100.5 |
1 : 36 | 0.86 | 0.84 | 101.9 |
Sample 5 | |||
1 : 2 | 14.87 | ||
1 : 3 | 10.07 | 9.91 | 101.6 |
1 : 4 | 7.50 | 7.43 | 100.8 |
1 : 12 | 2.69 | 2.48 | 108.4 |
1 : 36 | 0.99 | 0.83 | 119.4 |
Sample 6 | |||
1 : 2 | 13.16 | ||
1 : 3 | 8.96 | 8.77 | 102.2 |
1 : 4 | 6.78 | 6.58 | 103.0 |
1 : 12 | 2.27 | 2.19 | 103.4 |
1 : 36 | N.R. | N.R. | N.R. |
N.R.: not reported due to a technical issue.
*Value outside acceptance criteria.
Five quality controls (QCs) prepared with endogenous or spiked IL-13 in human plasma were selected at the lower limit of quantification (LLOQ), low (within 3 times the LLOQ), mid (midpoint on calibration curve), high (60–80% of the ULOQ), and upper limit of quantification (ULOQ) levels. QCs were run over three independent runs, three independent determinations per QC level. Nominal values for each QC was determined as the mean of the nine determinations performed and used as reference values for the inter- and intrarun accuracy and precision assessments.
Intrarun and interrun accuracy and precision were assessed using five independent determinations of each QC level over six validation runs. Each validation run was validated based on standard curve and the following acceptance criteria for QCs: accuracy of 70–130% from nominal value, CV ≤ 25%, and at least 4/5 determination of each QC passing the criteria. The intrarun accuracy and precision were calculated for each QC level as the mean accuracy and the precision of the five measurements per run. The interrun accuracy and precision were calculated for each QC level as the mean accuracy and precision of all measurements performed.
All three runs passed the acceptance criteria. The overall intrarun and interrun accuracy and precision were shown in Table
Sensitivity, working range, and intra- and interrun accuracy and precision of the IL-13 assay under acidic conditions. Inter- and intrarun accuracy and precision were determined based on five levels of QCs ranging from the LLOQ to the ULOQ, assessed over six runs and five preparations per run.
Parameters | Performance |
---|---|
Sensitivity | 0.59 pg/mL |
Working range | LLOQ: 0.59 pg/mL; ULOQ: 68.4 pg/mL |
Intrarun accuracy and precision | Accuracy: 84.6% to 116.2% |
Precision: 1.1% to 11.2% | |
Interrun accuracy and precision | Accuracy: 98.2 to 105.5% |
Precision: 5.0 to 12.6% |
The working range was defined by the LLOQ and ULOQ, whereby the sensitivity of the method was defined by the LLOQ (concentration at which the IL-13 concentration in human plasma could be quantitatively determined with acceptable precision and accuracy). The sensitivity of this IL-13 assay was 0.59 pg/mL in human plasma, taking into account the predilution of 1 : 2 required before analysis.
Three individual human plasmas were spiked with 100 ng/mL IL-13R alpha 2 and analyzed after dilution in sample diluent from 1 : 2, 1 : 3, and 1 : 4. The recovery of endogenous IL-13 level was within the range of 76.8 to 125.8% in the presence of rhIL-13R
The stability of IL-13 was investigated by analyzing two different lots of human plasma containing endogenous IL-13, stored undiluted under different storage conditions and time periods or subjected to 3 freeze (at −20 and −80°C for at least 24 hours) and thaw (at RT for at least 3 hours) cycles. Each stability sample was analyzed in duplicate and three independent preparations. Recovery was assessed against the concentration measured at Day 1 (T0). The results showed (Table
Stability assessment of endogenous IL-13 in human plasma. Two different human plasma samples (3.75 and 1.16 pg/mL of endogenous IL-13) were stored undiluted under different temperatures and time periods or subjected to freeze and thaw cycles. Recovery of IL-13 concentration was calculated using the concentration measured at Day 1 (T0) as a reference value.
Storage conditions | Mean recovery (%) | Range of recovery (%) | |
---|---|---|---|
Short-term stability | 25∘C, 1 week | 77.2 | 72.3–81.9 |
4∘C, 1 week | 82.3 | 74.0–94.3 | |
−20∘C, 1 month | 80.7 | 77.4–83.3 | |
−80∘C, 1 month | 76.1 | 73.9–78.2 | |
Freeze/thaw stability | 3 cycles at −20∘C | 92.2 | 73.6–112.5 |
3 cycles at −80∘C | 96.6 | 84.3–105.5 |
The assay was validated in human serum in a similar fashion as described for human plasma. The assay also demonstrated excellent performance, with a validated LLOQ of 0.27 pg/mL in serum, taking into account a minimum required dilution of 1 : 2. Intrarun accuracy and precision were in the range of 78.6–123.5% and 1.9–21.2%, respectively. Interrun accuracy and precision were in the range of 99.1–109.7% and 9.7–19.5%, respectively.
The validated IL-13 assay was applied to determine concentration of IL-13 in plasma samples from 30 atopic seasonal rhinitis patients and 34 healthy subjects. Endogenous IL-13 can be detected in 46.5% of human healthy subjects with a median of 0.5 pg/mL (values below the LLOQ were assigned as LLOQ/2 = 0.3 pg/mL), ranging from below limit of quantification to 12.99 pg/mL. IL-13 can also be detected in 40% of atopic patient samples with a median of 0.3 pg/mL (values below the LLOQ were assigned as LLOQ/2 = 0.3 pg/mL), with a range from below limit of quantification to 23.24 pg/mL. Taken altogether, the data demonstrated that the assay provides sensitive and reproducible measurement of IL-13 concentration in human plasma.
A strong overlap was observed over the ranges of distribution in the two subjects groups (Figure
IL-13 concentration in EDTA plasma of healthy donors and atopic seasonal rhinitis patients.
As many other biomarkers, IL-13 circulates at very low levels in blood, in the low pg/mL range. Besides, from our experience, the quantification of IL-13 at very low levels in complex matrices such as serum or plasma suffers from a lack of reproducibility that could be due to nonspecific binding and/or the presence of binding proteins. The study of the role of IL-13 in asthmatic and allergic diseases and the pharmacodynamic study of drugs in development require a sensitive and reproducible method of analysis. We herein described the development, validation, and implementation of a highly sensitive ELISA for the quantification of IL-13 in human serum and plasma. All reagents used in the assay were commercially available, including capture and detection monoclonal antibodies. The ELISA included a sample incubation step under strong acidic conditions (pH 3.5) that allowed to reduce nonspecific binding, to improve inter-assay reproducibility, and to reduce interference from binding proteins.
To the best of our knowledge, so far, only one report described the use of an ELISA combining an acidic sample treatment with an antigen-antibody binding step performed under strong acidic conditions. In this report, Lakshman et al. applied a similar acidic treatment to suppress the interference of myostatin binding proteins and therefore were able to measure total circulating levels of myostatin in serum [
pH influences protonation of ionizable amino acids on a protein, which are themselves involved in intraprotein, protein-solvent, protein-protein, and protein-ligand interactions [
It is possible that detection of IL-13 is partially inhibited at low pH; however, this effect should be compensated by the use of a calibration curve prepared in the same conditions (IL-13 in a buffer at low pH containing serum proteins to mimic human serum and plasma).
We could show that interference of IL-13R
The measurement of healthy and atopic subjects samples has shown that IL-13 can be quantified in about 40% of the samples. The quantified levels are consistent with data reported by Ledger et al. and Gauvreau et al., who used a bead-based immunoassay designed to quantify total IL-13 [
The sensitivity and validation data characterize this ELISA as a suitable method for the quantification of IL-13 in human plasma and human serum. Additional development and validation work could also allow to enlarge the method for the measurement of IL-13 in other matrices such as sputum, bronchoalveolar lavage fluid, or serum and plasma from preclinical species (mouse, rat, dog, or monkeys) if the interference of binding proteins is observed in these matrices. The use of this method could allow a better understanding of the implication of IL-13 in pathologies such as allergy or asthma and of the pharmacodynamic effect of new therapeutic drugs.
There are many other biomarkers for which soluble binding proteins have been identified, as for instance IL-1 [
Alkaline phosphatase
Bronchoalveolar lavage
Bovine serum albumin
Coefficient of variation
Fetal bovine serum
Horseradish peroxidase
Interleukin-13
Lower limit of quantification
Minimum required dilution
Nonspecific binding
Quality control
Room temperature
Upper limit of quantification.
(i) A novel sandwich ELISA for the quantitative and sensitive determination of IL-13 in human serum and plasma was established. (ii) The assay employs an incubation step at acidic pH, which was shown to decrease nonspecific binding and interference from IL-13 binding proteins. (iii) The assay was validated and was shown to be accurate and precise over the entire quantification range (0.59 to 68.4 pg/mL in human EDTA plasma). (iv) The validated assay was successfully applied to samples from healthy volunteers and patients with atopic seasonal rhinitis. (v) The assay is suitable for use in clinical trials to monitor efficacy or pharmacodynamic effects of drug candidates.