Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability methods
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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...
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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...
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