Ethical disclosure
Animal research
This study was carried out in accordance with the recommendations in the Guidelines for the Care and Use of Laboratory Animals of the Dutch Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of Maastricht. All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize suffering.
Human research
The recruitment of healthy volunteers was performed according to the Dutch Medical Ethical Committee (protocol: METC 11-3-056) and in respect of the Declaration of Helsinki. The individuals in this manuscript have given written informed consent to publish these case details.
Transverse Aortic Constricted (TAC) Mouse
Pressure overload was induced in male C57BL/6 J mice (Harlan Laboratories, Boxmeer, The Netherlands; N = 5) as previously described [9]. Control male C57BL/6 J mice (Sham; N = 5) underwent the same surgical procedure without the actual tightening of the ligature. Animals were sacrificed four weeks after the Sham or TAC operation and blood samples were collected.
LPS treated rats
Male Wistar rats (N = 5) were treated, under isoflurane anesthesia, with LPS (Sigma-Aldrich) (0.5 mg/kg) intravenously to induce an acute systemic inflammatory response, and were sacrificed after 30 minutes. For the semi-quantitative test serum from the animals was pooled. A total amount of 1000 μL was used in the membrane-based assay, while a total amount of 50 μL of serum per well was analyzed as required by the ELISA manufacturer’s instructions.
Sample preparation
Serum
Blood was sampled from the abdominal aorta during sacrifice. It was then allowed to clot for 4 hours at 4°C before centrifuging for 10 minutes at 2000 g. Serum was removed and immediately aliquoted and stored (for 6 months on average) at < -80°C.
Plasma
Blood was sampled from the abdominal aorta during sacrifice. Plasma was collected using EDTA as an anticoagulant. It was stored for 2 hours at 4°C and centrifuged for 10 minutes at approximately 2000 g. Plasma was removed and immediately aliquoted and stored (for 6 months on average) at < -80°C.
Membrane stripping
A pool of serum obtained from five TAC mice (200 μL for each) was tested according to the original Proteome Profiler™ Antibody Array based on chemiluminescence. Subsequently, stripping of the secondary antibody was performed by rinsing the membranes three times for 10 minutes with MilliQ water at 70°C. Then the new protocol for fluorescence detection, described in this manuscript (see below), was performed on the stripped membranes.
TNF-α detection via Enzyme Linked Immunosorbent Assay (ELISA)
Rat TNF-α Quantikine ELISA Kit was purchased from R& D Systems, Minneapolis MN, USA. The assay was performed following the manufacturer’s instructions.
Proteome Profiler™ Antibody Array: membrane-based assay
Mouse, rat and human Cytokine Array Panel A (R& D Systems) were used according to the manufacturer’s instructions (see the Extracellular Factors session on http://www.rndsystems.com/product_detail_objectname_ProteomeProfilerArray.aspx) until step #9 (Incubate overnight at 2 - 8°C on a rocking platform).
The following modifications of the original protocol were made to adapt it for fluorescence read-out:
-
1.
Carefully remove the membranes and place them into individual plastic containers. Gently wash the membranes with 20 mL of Wash Buffer 1× for 10 min on platform shaker. Repeat the procedure 3 times.
-
2.
Discard the washing buffer and rinse the membranes with 20 mL of blocking solution [Odyssey Blocking Buffer (LI-COR Bioscience, Lincoln NE, USA) and PBS 1× - (1:1)] for 15 minutes on a rocking platform.
-
3.
Every membrane is incubated with 5 mL blocking solution pre-mixed with 3.3 μL IRDye® 800CW Streptavidin (pre-diluted 1:100, LI-COR Bioscience) in 50 mL plastic tubes.
-
4.
Tubes are then placed for 1 hr on a rolling bench protected from the light.
-
5.
Wash the membranes 3 times with PBS 1× supplemented with 0.15%-Tween for 10 minutes.
-
6.
Rinse 4 times with PBS 1×.
-
7.
Leave the membranes to dry on paper in a dark place.
-
8.
Scan with the LI-COR Odyssey infrared imaging scanner (Westburg BV, Leusden, The Netherlands).
-
9.
Background subtraction was performed by setting the negative controls of each independent membrane to 0.
NB. The Odyssey imaging scanner adjusts the exposure time automatically upon the signal intensity set. It has default scanning presets. The user can choose from 5 settings through the “quality” option: Lowest, Low, Medium, High and Highest. The settings indicated under point 8 are therefore necessary to have the correct setup, allowing normalization of the data later on.
Standard curve with human recombinant proteins
Recombinant Human IL-1 beta and Recombinant Human CXCL10/IP-10 from R& D Systems (R& D Systems™) were reconstituted according to the manufacturer’s instructions. Following the reconstitution, the analytes were diluted to generate their respective stock solution.
The stock solutions were used to generate the following standard curves:
Concentrations (pg/mL): 1000, 500, 250,125, 62.5, 31.25.
Human IL-1 beta/IL-1 F2 Quantikine ELISA Kit and Human CXCL10/IP-10 Quantikine ELISA Kit (R& D Systems™) were used to quantify the analytes for further comparison with the Proteome Profiler™ outcome.
Statistical methods/data analysis
Mouse/rat data
All data included in this manuscript are expressed in units according to the international system. After background subtraction, polynomial curve fitting was used to infer the calibration curve for each membrane using Prism5 (GraphPad Software, Inc.; La Jolla CA, USA) and R [10]. Linear regression was used to test the co-linearity between ELISA and the membrane-based assay read-outs. The results are illustrated by bar and scatter plots.
Human data
Raw data have been imported in R. Background was subtracted from raw data for each membrane. The readouts of the membranes have been tested for a linear relationship with the predicted concentrations by use of the MASS package (‘Modern Applied Statistics with S’ (4th edition, 2002)). Study of the quality of predictions was performed by gvlma (Global Validation of Linear Models Assumptions).