Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability methods
Aim. Evidence-backed execution summary for Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability methods from Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability.
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This experiment, in seven questions
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Materials and Methods
reagent used in the protocol.
- Use
- Recombinant antigens were resuspended to 100 µg/mL in PBS with 0.1% BSA and pooled either in sub mixes of 10 or 20, or in a mix containing all of the antigens with the exception of CA125 and CA242 that were excluded due their origin in human cells/tissue and the resulting risk of contaminating proteins ( ). Ant...
Materials and Methods
reagent used in the protocol.
- Use
- Dilution series of bilirubin (Sigma-Aldrich) and intra-lipid (Fresenius Kabi, Uppsala Sweden) were prepared by making two-fold dilutions in H 2 O ranging from 200-6300 µg/mL and 3-200 mg/mL, respectively. When added to human serum samples the final concentrations were between 20-630 µg/mL...
Materials and Methods
reagent used in the protocol.
- Use
- Human serum samples were purchased from 3H Biological (Uppsala, Sweden).
Generation of PEA Probes
reagent used in the protocol.
- Use
- Antibodies, either matched monoclonal antibodies (mAb) or a polyclonal antibody (pAb) batch split in two, were resuspended at 1 mg/mL in PBS. A 40 kDa Zeba plate (Thermo Scientific, Rockford, IL) was equilibrated four times with 100 mM phosphate buffer, pH 7.3 (250 µL/well,1000 × g, 2 min). 20 µL antibo...
Generation of PEA Probes
reagent used in the protocol.
- Use
- Oligonucleotides synthesized with a 5′-thiol modification (Integrated DNA Technologies, Leuven, Belgium) were resuspended at 1 mM in 100 mM phosphate buffer with 20 mM EDTA, and distributed in a 96-well plate in 1.3 µL. 2.2 µL 40 mM DTT (Life Technologies, Eugene, OR) was added to 1.3 µL of each...
Generation of PEA Probes
reagent used in the protocol.
- Use
- The SMCC-treated antibodies were mixed with the DTT-treated oligonucleotides at 10x molar excess of oligo to Ab and transferred to pre-wet Slide-A-Lyzer Mini 7 MWCO dialysis cups (Thermo Scientific) and dialyzed in 1 L PBS with 5 mM EDTA at 4°C for two days with one buffer exchange to PBS. The PEA probes were f...
Proximity Extension Analysis
reagent used in the protocol.
- Use
- One µL sample (buffer (PBS with 0.1% BSA), antigen-spiked buffer, or biological sample) was mixed with 0.3 µL of each proximity probe mix (A and B), 0.3 µL Incubation Stabilizer (Olink Bioscience, Uppsala, Sweden) and 2.1 µL Incubation Solution (Olink Bioscience) and incubated overnight at 4°...
Internal Spike-in Controls
reagent used in the protocol.
- Use
- Internal spike-in controls included in the incubation buffer (incubation controls green fluorescent protein (GFP) and phycoerythrin (PE), extension control, and detection control) were individually titrated to reach threshold cycle (Cq)-values between 12 and 15 (for robust measurements) and pooled. Resulting concent...
Proximity Extension Analysis
One µL sample (buffer (PBS with 0.1% BSA), antigen-spiked buffer, or biological sample) was mixed with 0.3 µL of each proximity probe mix (A and B), 0.3 µL Incubation Stabilizer (Olink Bioscience, Uppsala, Sweden) and 2.1 µL Incubation Solution (Olink Bioscience) and incubated overnight at 4°...
- Use
- One µL sample (buffer (PBS with 0.1% BSA), antigen-spiked buffer, or biological sample) was mixed with 0.3 µL of each proximity probe mix (A and B), 0.3 µL Incubation Stabilizer (Olink Bioscience, Uppsala, Sweden) and 2.1 µL Incubation Solution (Olink Bioscience) and incubated overnight at 4°...
Internal Spike-in Controls
Internal spike-in controls included in the incubation buffer (incubation controls green fluorescent protein (GFP) and phycoerythrin (PE), extension control, and detection control) were individually titrated to reach threshold cycle (Cq)-values between 12 and 15 (for robust measurements) and pooled. Resulting concent...
- Use
- Internal spike-in controls included in the incubation buffer (incubation controls green fluorescent protein (GFP) and phycoerythrin (PE), extension control, and detection control) were individually titrated to reach threshold cycle (Cq)-values between 12 and 15 (for robust measurements) and pooled. Resulting concent...
Xenograft Experiments
Three-to-four-week-old female NMRI-nude mice (Taconic, Denmark) were housed in individually ventilated cages at a research animal facility at Uppsala University (Uppsala, Sweden) according to regulations. Human neuroblastoma cells (SK-N-FI) were mixed at a 1∶1 volume ratio with Matrigel (BD Biosciences, San Di...
- Use
- Three-to-four-week-old female NMRI-nude mice (Taconic, Denmark) were housed in individually ventilated cages at a research animal facility at Uppsala University (Uppsala, Sweden) according to regulations. Human neuroblastoma cells (SK-N-FI) were mixed at a 1∶1 volume ratio with Matrigel (BD Biosciences, San Di...
Improved Preamplification Protocol Resulting in Increased Precision and Sensitivity
As the readout is performed with a microfluidic qPCR system that measures only 7 nL reactions, a preamplification step had to be performed to reach sufficient signal. A rule of thumb, according to Fluidigm's guide lines, is to use samples with sufficient numbers of templates to get a Cq-value <25 for good prec...
- Use
- As the readout is performed with a microfluidic qPCR system that measures only 7 nL reactions, a preamplification step had to be performed to reach sufficient signal. A rule of thumb, according to Fluidigm's guide lines, is to use samples with sufficient numbers of templates to get a Cq-value <25 for good prec...
Demonstrating High Specificity and Scalability of PEA
Multiplexing of immunoassays is generally constrained because of false positive signals generated by unspecific binding of antibodies. One of the benefits of PEA and other proximity probing techniques is the high specificity. This is brought about by two things: proximal requirement of the antibodies to generate a s...
- Use
- Multiplexing of immunoassays is generally constrained because of false positive signals generated by unspecific binding of antibodies. One of the benefits of PEA and other proximity probing techniques is the high specificity. This is brought about by two things: proximal requirement of the antibodies to generate a s...
Demonstrating High Specificity and Scalability of PEA
Furthermore, the response detected for entire antigen pool was similar to that of the antigen sub mix ( ). On average, the difference in ddCq between the mix containing all antigens and each sub mix was as low as 0.1 (equivalent to a 7% change in signal), and with the highest difference observed for adrenomedullin a...
- Use
- Furthermore, the response detected for entire antigen pool was similar to that of the antigen sub mix ( ). On average, the difference in ddCq between the mix containing all antigens and each sub mix was as low as 0.1 (equivalent to a 7% change in signal), and with the highest difference observed for adrenomedullin a...
Function and Lack of Cross-reactivity in Biological Samples
Normal and pathological human plasma samples contain a number of endogenous interfering substances, such as heterophilic antibodies possessing broad reactivity with antibodies of other animal species, and are therefore likely to hamper the performance of an immunoassay. To evaluate the potential impact of this spec...
- Use
- Normal and pathological human plasma samples contain a number of endogenous interfering substances, such as heterophilic antibodies possessing broad reactivity with antibodies of other animal species, and are therefore likely to hamper the performance of an immunoassay. To evaluate the potential impact of this spec...
Before you run
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First confirmation
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Ethics Statement
The Uppsala Animal Ethics Committee has approved the animal xenograft studies (ID number C215/12), and all guidelines were followed. The mice were inspected every day and tumor sizes were measured every second to third day. The mice were sacrificed after blood collection by controlled exposure to increasing concentration of CO 2. After the mice had died cervical dislocation was performed as an extra measure of security. It can therefore not be considered as a standard survival study since the experiment was terminated before any mouse's health was put in danger. No animal dies as a result of the intervention. According to the research animal permit a mouse has to be euthanized if the subcutaneous tumor reach a size above 10 mm in diameter or if the tumor gets ulcerous. This was never the case in this study. Anesthetics is not common use for subcutaneous tumor cell injection or...
Materials and Methods
Recombinant antigens were resuspended to 100 µg/mL in PBS with 0.1% BSA and pooled either in sub mixes of 10 or 20, or in a mix containing all of the antigens with the exception of CA125 and CA242 that were excluded due their origin in human cells/tissue and the resulting risk of contaminating proteins ( ). Antibodies, either matched monoclonal antibodies (mAb) or a polyclonal antibody (pAb) batch split in two, were resuspended at 1 mg/mL in PBS. All antibodies were purchased from commercial sources. For the IL-6 assays used for and, the following antibody (Ab) combinations were used: A mAb/B mAb: A = no. MAB206, clone 6708 (RnD Systems), B = no. 554541, clone MQ2-13A5 (BD Pharmingen); A mAb/B pAb: A = no. MAB206, clone 6708, B = no. AF-206-NA (RnD Systems); A pAb/B mAb: A = no. AF-206-NA, B = no. 554541, cl...
Materials and Methods
The hyper-thermostable DNA polymerases; Pwo Hypernova (DNA Gdansk), Pfu (Thermo Fisher Scientific Inc., Waltham, MA), TLA (Bioneer, Daejeon, South Korea), Pwo Delta3 (DNA Gdansk), KOD exo+, KOD exo- (Toyobo, Osaka, Japan), and DreamTaq (Thermo Fisher Scientific Inc) were evaluated at 0.5 units per reaction with the following thermocycling protocol: 10 min room temperature (RT) incubation, 23 min extension at RT/37°C/45°C, 10 min denaturation at 85°C.
Materials and Methods
Dilution series of bilirubin (Sigma-Aldrich) and intra-lipid (Fresenius Kabi, Uppsala Sweden) were prepared by making two-fold dilutions in H 2 O ranging from 200-6300 µg/mL and 3-200 mg/mL, respectively. When added to human serum samples the final concentrations were between 20-630 µg/mL bilirubin and 0.3-20 mg/mL intralipid. Hemolysate samples were prepared at final concentrations ranging from 0.23-15 g/L, where 15 g/L corresponds to complete lysis of 10% of all blood cells in a sample. Two whole heparinized blood samples (27 mL at Hb 159 g/L) were spun in 50 ml Falcon tubes at 3000 rpm, 10 min at 10°C, without brake. Cell pellets were washed four times with 0.9% NaCl, resuspended in H 2 O to the original volume, freeze-thawed, vigorously mixed four times, pooled, and spun 5000 rpm, 20 min at 10°C. The final Hb-value in the hemolysat...
Generation of PEA Probes
Antibodies, either matched monoclonal antibodies (mAb) or a polyclonal antibody (pAb) batch split in two, were resuspended at 1 mg/mL in PBS. A 40 kDa Zeba plate (Thermo Scientific, Rockford, IL) was equilibrated four times with 100 mM phosphate buffer, pH 7.3 (250 µL/well,1000 × g, 2 min). 20 µL antibody was added/well, spun 1000 × g, 3 min, and collected in a new plate. Sulfo-SMCC (Thermo Scientific) was dissolved to 3.33 mM in 100 mM phosphate buffer. 2 µL sulfo-SMCC was added to each antibody well, mixed, and incubated at 4°C for 2 h with three times intermittent mixing. A new 40 kDa Zeba plate was equilibrated with 100 mM phosphate buffer with 20 mM EDTA. The SMCC-treated antibodies were transferred to the Zeba plate and spun at 1000 × g, 3 min. Conjugation protocols were previously optimized to function well on both mAb and pAb.
Generation of PEA Probes
Oligonucleotides synthesized with a 5′-thiol modification (Integrated DNA Technologies, Leuven, Belgium) were resuspended at 1 mM in 100 mM phosphate buffer with 20 mM EDTA, and distributed in a 96-well plate in 1.3 µL. 2.2 µL 40 mM DTT (Life Technologies, Eugene, OR) was added to 1.3 µL of each oligonucleotide, mixed, and incubated at 95°C, 2 min, followed by a 1 h incubation step at 37°C. 20 µL PBS buffer with 20 mM EDTA was added to the oligonucleotides and excess DTT was removed using two consecutive 7 kDa Zeba plates equilibrated with 100 mM phosphate buffer.
Generation of PEA Probes
The SMCC-treated antibodies were mixed with the DTT-treated oligonucleotides at 10x molar excess of oligo to Ab and transferred to pre-wet Slide-A-Lyzer Mini 7 MWCO dialysis cups (Thermo Scientific) and dialyzed in 1 L PBS with 5 mM EDTA at 4°C for two days with one buffer exchange to PBS. The PEA probes were finally diluted to 75 µg/mL in a PBS buffer containing 4 mM EDTA, 35 µg/mL ssDNA (Sigma-Aldrich), 0.1% fish gelatin, and 20 mM Tris HCl, and stored at 4°C.
Proximity Extension Analysis
One µL sample (buffer (PBS with 0.1% BSA), antigen-spiked buffer, or biological sample) was mixed with 0.3 µL of each proximity probe mix (A and B), 0.3 µL Incubation Stabilizer (Olink Bioscience, Uppsala, Sweden) and 2.1 µL Incubation Solution (Olink Bioscience) and incubated overnight at 4°C ( ). A combined extension and preamplification mix (96 µL) containing 10 µL MUX PEA Solution (Olink Bioscience), 0.5 units Pwo (DNA Gdansk, Poland), 1 µM forward + reverse universal preamplification primers, and 1 unit hot-start DNA polymerase was added to each reaction at RT. After mixing and a total 5-min incubation, the plate was transferred to a thermocycler (Applied Biosystem 2720) running an initial extension step to unite the two oligonucleotides (50°C, 20 min), immediately followed by a hot-start step (95°C, 5 min) and 17 cycles of amplif...
Measurement outputs
What raw and processed outputs should exist?
(A) 94 pairs of specific antibodies are equipped with oligonucletotides (PEA probes) and mixed with an antigen-containinig sample. (B) Upon sample incubation, all proximity prob...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
One µL sample (buffer (PBS with 0.1% BSA), antigen-spiked buffer, or biological sample) was mixed with 0.3 µL of each proximity probe mix (A and B), 0.3 µL Incuba...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Blood was sampled by venipuncture and transferred into EDTA sample tubes. Tubes were inverted 10x directly after sampling. Tubes were stored and transported in an upright positi...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
For each data point, the raw Cq-value (log 2 scale) was normalized by subtracting the Cq-value for the extension control reaction for the corresponding sample, thereby correctin...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
To evaluate the specificity of the PEA, the 90 antigens where divided into eight sub mixes comprising assays (by sequence ID) 1-11, 12-22, 23-32, 33-42, 43-54, 55-64, 65-74, and 75-96, respectively ( ).
from paperScoring or quantification
Quantify the primary readouts for this experiment: (A) 94 pairs of specific antibodies are equipped with oligonucletotides (PEA probes) and mixed with an antigen-containinig sample. (B) Upon sample incubation, all proximity prob...; One µL sample (buffer (PBS with 0.1% BSA), antigen-spiked buffer, or biological sample) was mixed with 0.3 µL of each proximity probe mix (A and B), 0.3 µL Incuba...; Blood was sampled by venipuncture and transferred into EDTA sample tubes. Tubes were inverted 10x directly after sampling. Tubes were stored and transported in an upright positi...; For each data point, the raw Cq-value (log 2 scale) was normalized by subtracting the Cq-value for the extension control reaction for the corresponding sample, thereby correctin....
from paperStatistical comparison
To evaluate the specificity of the PEA, the 90 antigens where divided into eight sub mixes comprising assays (by sequence ID) 1-11, 12-22, 23-32, 33-42,...; Nude mice were grafted with human tumor cells (SK-N-FI). Before (white symbols) and on day 30 after inoculation (gray symbols), the sera were analyzed with 96-plex PEA and compa...; Other known serum and plasma components known to affect immunoassay performance are hemolysate, lipids, and bilirubin. Serial dilutions of these components were performed and ad...
from paperReporting output
Report representative outputs alongside summary comparisons for (A) 94 pairs of specific antibodies are equipped with oligonucletotides (PEA probes) and mixed with an antigen-containinig sample. (B) Upon sample incubation, all proximity prob..., One µL sample (buffer (PBS with 0.1% BSA), antigen-spiked buffer, or biological sample) was mixed with 0.3 µL of each proximity probe mix (A and B), 0.3 µL Incuba..., Blood was sampled by venipuncture and transferred into EDTA sample tubes. Tubes were inverted 10x directly after sampling. Tubes were stored and transported in an upright positi..., For each data point, the raw Cq-value (log 2 scale) was normalized by subtracting the Cq-value for the extension control reaction for the corresponding sample, thereby correctin....
inferred from protocolStructured statistical methods
To evaluate the specificity of the PEA, the 90 antigens where divided into eight sub mixes comprising assays (by sequence ID) 1-11, 12-22, 23-32, 33-42,...; Nude mice were grafted with human tumor cells (SK-N-FI). Before (white symbols) and on day 30 after inoculation (gray symbols), the sera were analyzed with 96-plex PEA and compa...; Other known serum and plasma components known to affect immunoassay performance are hemolysate, lipids, and bilirubin. Serial dilutions of these components were performed and ad...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
The Uppsala Animal Ethics Committee has approved the animal xenograft studies (ID number C215/12), and all guidelines were followed. The mice were inspected every day and tumor sizes were measured every second to third day. The mice were sacrificed after blood collection by controlled exposure to increasing concentration of CO 2. After the mice had died cervical dislocation was performed as an extra measure of security. It can therefore not be considered as a standard survival study since the experiment was terminated before any mouse's health was put in danger. No animal dies as a result of the intervention. According to the research animal permit a mouse has to be euthanized if the subcutaneous tumor reach a size above 10 mm in diameter or if the tumor gets ulcerous. This was never the case in this study. Anesthetics is not common use for subcutaneous tumor cell injection or blood sampling. It was therefore not used.
Recombinant antigens were resuspended to 100 µg/mL in PBS with 0.1% BSA and pooled either in sub mixes of 10 or 20, or in a mix containing all of the antigens with the exception of CA125 and CA242 that were excluded due their origin in human cells/tissue and the resulting risk of contaminating proteins ( ). Antibodies, either matched monoclonal antibodies (mAb) or a polyclonal antibody (pAb) batch split in two, were resuspended at 1 mg/mL in PBS. All antibodies were purchased from commercial sources. For the IL-6 assays used for and, the following antibody (Ab) combinations were used: A mAb/B mAb: A = no. MAB206, clone 6708 (RnD Systems), B = no. 554541, clone MQ2-13A5 (BD Pharmingen); A mAb/B pAb: A = no. MAB206, clone 6708, B = no. AF-206-NA (RnD Systems); A pAb/B mAb: A = no. AF-206-NA, B = no. 554541, clone MQ2-13A5; A pAb/B pAb: A/B = no. AF-206-NA.
The hyper-thermostable DNA polymerases; Pwo Hypernova (DNA Gdansk), Pfu (Thermo Fisher Scientific Inc., Waltham, MA), TLA (Bioneer, Daejeon, South Korea), Pwo Delta3 (DNA Gdansk), KOD exo+, KOD exo- (Toyobo, Osaka, Japan), and DreamTaq (Thermo Fisher Scientific Inc) were evaluated at 0.5 units per reaction with the following thermocycling protocol: 10 min room temperature (RT) incubation, 23 min extension at RT/37°C/45°C, 10 min denaturation at 85°C.
Dilution series of bilirubin (Sigma-Aldrich) and intra-lipid (Fresenius Kabi, Uppsala Sweden) were prepared by making two-fold dilutions in H 2 O ranging from 200-6300 µg/mL and 3-200 mg/mL, respectively. When added to human serum samples the final concentrations were between 20-630 µg/mL bilirubin and 0.3-20 mg/mL intralipid. Hemolysate samples were prepared at final concentrations ranging from 0.23-15 g/L, where 15 g/L corresponds to complete lysis of 10% of all blood cells in a sample. Two whole heparinized blood samples (27 mL at Hb 159 g/L) were spun in 50 ml Falcon tubes at 3000 rpm, 10 min at 10°C, without brake. Cell pellets were washed four times with 0.9% NaCl, resuspended in H 2 O to the original volume, freeze-thawed, vigorously mixed four times, pooled, and spun 5000 rpm, 20 min at 10°C. The final Hb-value in the hemolysate stock solution was estimated to 150 g/L.
Antibodies, either matched monoclonal antibodies (mAb) or a polyclonal antibody (pAb) batch split in two, were resuspended at 1 mg/mL in PBS. A 40 kDa Zeba plate (Thermo Scientific, Rockford, IL) was equilibrated four times with 100 mM phosphate buffer, pH 7.3 (250 µL/well,1000 × g, 2 min). 20 µL antibody was added/well, spun 1000 × g, 3 min, and collected in a new plate. Sulfo-SMCC (Thermo Scientific) was dissolved to 3.33 mM in 100 mM phosphate buffer. 2 µL sulfo-SMCC was added to each antibody well, mixed, and incubated at 4°C for 2 h with three times intermittent mixing. A new 40 kDa Zeba plate was equilibrated with 100 mM phosphate buffer with 20 mM EDTA. The SMCC-treated antibodies were transferred to the Zeba plate and spun at 1000 × g, 3 min. Conjugation protocols were previously optimized to function well on both mAb and pAb.
Oligonucleotides synthesized with a 5′-thiol modification (Integrated DNA Technologies, Leuven, Belgium) were resuspended at 1 mM in 100 mM phosphate buffer with 20 mM EDTA, and distributed in a 96-well plate in 1.3 µL. 2.2 µL 40 mM DTT (Life Technologies, Eugene, OR) was added to 1.3 µL of each oligonucleotide, mixed, and incubated at 95°C, 2 min, followed by a 1 h incubation step at 37°C. 20 µL PBS buffer with 20 mM EDTA was added to the oligonucleotides and excess DTT was removed using two consecutive 7 kDa Zeba plates equilibrated with 100 mM phosphate buffer.
The SMCC-treated antibodies were mixed with the DTT-treated oligonucleotides at 10x molar excess of oligo to Ab and transferred to pre-wet Slide-A-Lyzer Mini 7 MWCO dialysis cups (Thermo Scientific) and dialyzed in 1 L PBS with 5 mM EDTA at 4°C for two days with one buffer exchange to PBS. The PEA probes were finally diluted to 75 µg/mL in a PBS buffer containing 4 mM EDTA, 35 µg/mL ssDNA (Sigma-Aldrich), 0.1% fish gelatin, and 20 mM Tris HCl, and stored at 4°C.
One µL sample (buffer (PBS with 0.1% BSA), antigen-spiked buffer, or biological sample) was mixed with 0.3 µL of each proximity probe mix (A and B), 0.3 µL Incubation Stabilizer (Olink Bioscience, Uppsala, Sweden) and 2.1 µL Incubation Solution (Olink Bioscience) and incubated overnight at 4°C ( ). A combined extension and preamplification mix (96 µL) containing 10 µL MUX PEA Solution (Olink Bioscience), 0.5 units Pwo (DNA Gdansk, Poland), 1 µM forward + reverse universal preamplification primers, and 1 unit hot-start DNA polymerase was added to each reaction at RT. After mixing and a total 5-min incubation, the plate was transferred to a thermocycler (Applied Biosystem 2720) running an initial extension step to unite the two oligonucleotides (50°C, 20 min), immediately followed by a hot-start step (95°C, 5 min) and 17 cycles of amplification (95°C, 30 s; 54°C, 1 min; 60°C, 1 min) ( ). Amplification was performed with universal flanking primers to amplify all 96 sequences in parallel ( ). Finally, 2.8 µL of the preamplification products were mixed with 7.2 µL buffer containing 5 µL MUX Detection Solu...
Machine-readable layer
[
{
"@context": "https://schema.org",
"@type": "HowTo",
"name": "Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability methods",
"description": "Evidence-backed execution summary for Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability methods from Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability.",
"totalTime": "PT44M",
"step": [
{
"@type": "HowToStep",
"position": 1,
"name": "Ethics Statement",
"text": "The Uppsala Animal Ethics Committee has approved the animal xenograft studies (ID number C215/12), and all guidelines were followed. The mice were inspected every day and tumor sizes were measured every second to third day. The mice were sacrificed after blood collection by controlled exposure to increasing concentration of CO 2. After the mice had died cervical dislocation was performed as an extra measure of security. It can therefore not be considered as a standard survival study since the experiment was terminated before any mouse's health was put in danger. No animal dies as a result of the intervention. According to the research animal permit a mouse has to be euthanized if the subcutaneous tumor reach a size above 10 mm in diameter or if the tumor gets ulcerous. This was never the case in this study. Anesthetics is not common use for subcutaneous tumor cell injection or..."
},
{
"@type": "HowToStep",
"position": 2,
"name": "Materials and Methods",
"text": "Recombinant antigens were resuspended to 100 µg/mL in PBS with 0.1% BSA and pooled either in sub mixes of 10 or 20, or in a mix containing all of the antigens with the exception of CA125 and CA242 that were excluded due their origin in human cells/tissue and the resulting risk of contaminating proteins ( ). Antibodies, either matched monoclonal antibodies (mAb) or a polyclonal antibody (pAb) batch split in two, were resuspended at 1 mg/mL in PBS. All antibodies were purchased from commercial sources. For the IL-6 assays used for and, the following antibody (Ab) combinations were used: A mAb/B mAb: A = no. MAB206, clone 6708 (RnD Systems), B = no. 554541, clone MQ2-13A5 (BD Pharmingen); A mAb/B pAb: A = no. MAB206, clone 6708, B = no. AF-206-NA (RnD Systems); A pAb/B mAb: A = no. AF-206-NA, B = no. 554541, cl..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Materials and Methods",
"text": "The hyper-thermostable DNA polymerases; Pwo Hypernova (DNA Gdansk), Pfu (Thermo Fisher Scientific Inc., Waltham, MA), TLA (Bioneer, Daejeon, South Korea), Pwo Delta3 (DNA Gdansk), KOD exo+, KOD exo- (Toyobo, Osaka, Japan), and DreamTaq (Thermo Fisher Scientific Inc) were evaluated at 0.5 units per reaction with the following thermocycling protocol: 10 min room temperature (RT) incubation, 23 min extension at RT/37°C/45°C, 10 min denaturation at 85°C."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Materials and Methods",
"text": "Dilution series of bilirubin (Sigma-Aldrich) and intra-lipid (Fresenius Kabi, Uppsala Sweden) were prepared by making two-fold dilutions in H 2 O ranging from 200-6300 µg/mL and 3-200 mg/mL, respectively. When added to human serum samples the final concentrations were between 20-630 µg/mL bilirubin and 0.3-20 mg/mL intralipid. Hemolysate samples were prepared at final concentrations ranging from 0.23-15 g/L, where 15 g/L corresponds to complete lysis of 10% of all blood cells in a sample. Two whole heparinized blood samples (27 mL at Hb 159 g/L) were spun in 50 ml Falcon tubes at 3000 rpm, 10 min at 10°C, without brake. Cell pellets were washed four times with 0.9% NaCl, resuspended in H 2 O to the original volume, freeze-thawed, vigorously mixed four times, pooled, and spun 5000 rpm, 20 min at 10°C. The final Hb-value in the hemolysat..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Generation of PEA Probes",
"text": "Antibodies, either matched monoclonal antibodies (mAb) or a polyclonal antibody (pAb) batch split in two, were resuspended at 1 mg/mL in PBS. A 40 kDa Zeba plate (Thermo Scientific, Rockford, IL) was equilibrated four times with 100 mM phosphate buffer, pH 7.3 (250 µL/well,1000 × g, 2 min). 20 µL antibody was added/well, spun 1000 × g, 3 min, and collected in a new plate. Sulfo-SMCC (Thermo Scientific) was dissolved to 3.33 mM in 100 mM phosphate buffer. 2 µL sulfo-SMCC was added to each antibody well, mixed, and incubated at 4°C for 2 h with three times intermittent mixing. A new 40 kDa Zeba plate was equilibrated with 100 mM phosphate buffer with 20 mM EDTA. The SMCC-treated antibodies were transferred to the Zeba plate and spun at 1000 × g, 3 min. Conjugation protocols were previously optimized to function well on both mAb and pAb."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Generation of PEA Probes",
"text": "Oligonucleotides synthesized with a 5′-thiol modification (Integrated DNA Technologies, Leuven, Belgium) were resuspended at 1 mM in 100 mM phosphate buffer with 20 mM EDTA, and distributed in a 96-well plate in 1.3 µL. 2.2 µL 40 mM DTT (Life Technologies, Eugene, OR) was added to 1.3 µL of each oligonucleotide, mixed, and incubated at 95°C, 2 min, followed by a 1 h incubation step at 37°C. 20 µL PBS buffer with 20 mM EDTA was added to the oligonucleotides and excess DTT was removed using two consecutive 7 kDa Zeba plates equilibrated with 100 mM phosphate buffer."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Generation of PEA Probes",
"text": "The SMCC-treated antibodies were mixed with the DTT-treated oligonucleotides at 10x molar excess of oligo to Ab and transferred to pre-wet Slide-A-Lyzer Mini 7 MWCO dialysis cups (Thermo Scientific) and dialyzed in 1 L PBS with 5 mM EDTA at 4°C for two days with one buffer exchange to PBS. The PEA probes were finally diluted to 75 µg/mL in a PBS buffer containing 4 mM EDTA, 35 µg/mL ssDNA (Sigma-Aldrich), 0.1% fish gelatin, and 20 mM Tris HCl, and stored at 4°C."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Proximity Extension Analysis",
"text": "One µL sample (buffer (PBS with 0.1% BSA), antigen-spiked buffer, or biological sample) was mixed with 0.3 µL of each proximity probe mix (A and B), 0.3 µL Incubation Stabilizer (Olink Bioscience, Uppsala, Sweden) and 2.1 µL Incubation Solution (Olink Bioscience) and incubated overnight at 4°C ( ). A combined extension and preamplification mix (96 µL) containing 10 µL MUX PEA Solution (Olink Bioscience), 0.5 units Pwo (DNA Gdansk, Poland), 1 µM forward + reverse universal preamplification primers, and 1 unit hot-start DNA polymerase was added to each reaction at RT. After mixing and a total 5-min incubation, the plate was transferred to a thermocycler (Applied Biosystem 2720) running an initial extension step to unite the two oligonucleotides (50°C, 20 min), immediately followed by a hot-start step (95°C, 5 min) and 17 cycles of amplif..."
}
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{
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"name": "Xenograft Experiments"
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{
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"name": "Demonstrating High Specificity and Scalability of PEA"
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{
"@type": "HowToTool",
"name": "Demonstrating High Specificity and Scalability of PEA"
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{
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"@type": "HowToSupply",
"name": "Generation of PEA Probes"
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"headline": "Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability",
"datePublished": "2014",
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"name": "Erika Assarsson"
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