Low volume, highly sensitive immunoassays for detecting cytokines in animal fluids Jean-Francois Michaud, Nathalie Rouleau and Francesco Lipari Cytokine levels are measured in many different experimental models, with samples obtained in a variety of biological matrices such as cell culture supernatant, serum, plasma, saliva, cerebrospinal fluid, and tissue homogenate. The most common method of cytokine analysis is enzyme-linked immunosorbent assay (ELISA). However, ELISA often requires high sample volume (at least 50 µL), which is difficult to obtain when working with small animals such as mice. Also, the ELISA method has a limited dynamic range and low throughput due to its numerous wash steps. Considering the limitations of ELISA technology, a faster approach was developed for cytokine analysis in complex biological samples. This novel analytical approach is a chemiluminescent homogeneous bead-based immunoassay that is performed without wash steps. The cytokine of interest is sandwiched between two highly specific antibodies bound to the beads. In presence of the analyte, the beads come into proximity resulting in an emission of light upon laser excitation. The amount of light emitted is directly proportional to the amount of analyte present. Sample volumes as low as 5 µL can be utilized in this sensitive detection platform. In addition, upper quantitation limits can extend to much higher concentrations than achievable using traditional ELISA assay. For example, mouse IL6 can be detected from 1 pg/mL to 100,000 pg/mL without dilution, whereas a comparable ELISA has a range of 8 pg/mL to 500 pg/mL. The application of this immunoassay to different types of biological samples was verified by performing spike recovery experiments, and also by comparison of the levels measured to the levels reported in the literature. Overall, this assay technology will aid researchers in evaluating cytokines from many different biological sources that, in some cases, have been problematic to measure in the past. 3 Tested diluent X X X Assay buffer from kit X X X HiBlock buffer X X X PBS-0.1%BSA X X X PBS-0.01%BSA X X X Same fluid as sample fluid, but other species X FBS* *For serum samples, FBS was the only diluent tested, as FBS has previously been shown to be the best diluent for all serum samples (data not shown). NaCl buffer The biotinylated anti-analyte antibody binds to the Streptavidin-coated Donor beads while another anti-analyte antibody is conjugated to AlphaLISA Acceptor beads. In the presence of the analyte, the beads come into close proximity. The excitation of the Donor beads provokes the release of singlet oxygen molecules that triggers a cascade of energy transfer in the Acceptor beads resulting in a sharp peak of light emission at 615 nm. 4 With step 1 results, select the two best diluents based on: • Observed concentration X dilution factor (DF) as close as possible to spiked value, for all dilution points • Recoveries between 70-130% for all dilution points Immunoassays – quick and simple Spike neat and ½ diluted mouse/rat fluid with mouse cytokine Use spiked diluent as spike control Select 3-4 spikes and include a “no spike” control (to measure physiological level) Recoveries between 70-130% are targeted Evaluate other diluents if good recovery is not obtained Assay protocol with 3 incubation steps (No wash steps) 5.0 5.0 5.0 5.0 3.0 3.0 3.0 3.0 3.0 2.0 2.0 2.0 2.0 2.0 1.0 1.0 1.0 1.0 1.0 0.0 0.0 0.0 0.0 r2 neat = 0.8832 2 4.0 r dil 1/2 = 0.9995 0.0 0.0 0.5 1.0 7 Example (no dilution required) Step 1: Linearity of dilutions in 5 diluents for spiked mouse BALF (3ng/mL mTNFα) Add 10 L of AlphaLISA Anti-Analyte Acceptor beads Add 10 L of Biotinylated Anti-Analyte Antibody Incubate 60 minutes AlphaLISA buffer 5.0 Mouse/rat sample fluids: All mouse/rat sample fluids were supplied as pools of individuals, nonmedicated, non-immunized, from Bioreclamation LLC: • Mouse bronchial lavage fluid (BALF): Strain CD-1, cat# MSE-BRONLEV • Mouse serum: Strain BALB/C, cat# MSESRM-BALB • Rat amniotic fluid (AF): Strain Sprague Dawley, cat# RATAMNFL • Rat cerebrospinal fluid (CSF): Strain Sprague Dawley, cat# RATCSF 5 AlphaLISA Signal (counts) Calibration curve for mouse MCP-1 (LDL = Lower Detection Limit) 1,000,000 LDL = 2.4 pg/mL 100,000 10,000 5.0 r2 neat = 0.9905 HiBlock buffer r2 neat = 0.9906 PBS - 0.1%BSA 5.0 r2 neat = 0.9993 PBS - 0.01%BSA 5.0 r2 neat = 0.9998 Beagle BALF 5.0 r2 neat = 0.9977 4.0 4.0 4.0 4.0 4.0 3.0 3.0 3.0 3.0 3.0 2.0 2.0 2.0 2.0 2.0 1.0 1.0 1.0 1.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 1.0 Dilution Incubate 30 minutes Read at 615 nm using Envision® or EnSpireTM Reader and analyze data 100 - -12 -11 -10 -9 -8 -7 -6 Log [mouse MCP-1] (g/mL) Fluids and cytokines chosen 0.5 1.0 Dilution 0.5 1.0 PBS-0.1%BSA 0.5 1.0 Dilution Dilution AlphaLISA buffer HiBlock buffer PBS-0.01%BSA 0.5 Physiological range found in literature (pg/mL) Cytokine Mouse serum Mouse BALF Rat CSF Rat AF IL6 5 - 70 20 Not found 10 - 60 TNFα 5 - 100 30-90 Not found 10 - 60 MCP-1 30 - 1500 40 Not found 1500 1 2 4 8 16 32 100 179 187 180 170 192 0.5 1.0 PBS-0.01%BSA Recovery from 1/2 dil. (%) Recovery from neat (%) 100 104 100 95 107 100 127 131 129 129 129 0.5 1.0 r2 neat = 0.9496 4.0 r2 dil 1/2 = 0.9998 0.0 0.0 0.5 Dilution Dilution Recovery from 1/2 dil. (%) Recovery from neat (%) 100 103 101 101 101 100 144 157 154 163 169 1.0 r2 neat = 0.9935 4.0 r2 dil 1/2 = 0.9974 0.0 0.0 0.5 PBS-0.1%BSA Recovery from 1/2 dil. (%) Recovery from neat (%) 100 109 107 113 117 100 149 152 145 150 148 1.0 Dilution Dilution HiBlock buffer Diluents not chosen: observed concentration X DF not close to spiked value (3ng/mL) (too low or too high). Rabbit AF Recovery from 1/2 dil. (%) Recovery from neat (%) Recovery from 1/2 dil. (%) 100 102 98 100 100 100 114 126 123 116 119 100 110 108 102 104 Choose 2 good diluents: PBS-0.1%BSA and rabbit AF • Excellent linearity from ½ dilution • Observed concentration X DF close to spiked value (3ng/mL) for all dilutions. Step 3: Chosen diluent showing the best spike and recovery results: Dilution Step 3: Chosen diluent showing the best spike and recovery results No spike 10 30 300 3000 Cytokine Beagle BALF Choose 2 good diluents: PBS-0.1%BSA and Beagle BALF • Good linearity from neat • Observed concentration X DF close to spiked value (3ng/mL) for all dilutions Diluent: PBS-0.1%BSA Spiked diluent Spiked neat Mouse BALF control Concentration Concentration Recovery (%)** (pg/mL)* (pg/mL)* 0.0 17.1 NA 12.0 9.2 76 35.2 32.3 92 300.2 292.6 97 3141.2 2952.1 94 Excellent linearity of dilutions from neat (Step 1) and excellent recovery for all 4 spikes tested. Measured TNFα physiological level in mouse BALF (No spike) = 17.1 pg/mL * Concentration for 10-3000 pg/mL spikes = measured concentration – no spike value. ** Recovery (%) = recovery compared to spiked diluent control Spiked 1/2 Rat AF Concentration (pg/mL)* 575.6 317.2 750.4 2227.9 Recovery (%)** NA 119 84 85 ½ dilution in diluent required: excellent recovery for all 3 spikes tested in ½ rat AF, but not in neat rat AF. Measured MCP-1 physiological level in rat AF (No spike) = 575.6 pg/mL X 2 = 1151pg/mL * Concentration for 300-3000 pg/mL spikes = measured concentration – no spike value. ** Recovery (%) = recovery compared to spiked diluent control IL6 TNFα MCP-1 Diluents not chosen: Linearity not good from neat Spike (pg/mL) Spiked Spiked neat Rat AF diluent control Concentration Concentration Recovery Spike (pg/mL) (pg/mL)* (pg/mL)* (%)** No spike 0.0 910.5 NA 300 267.3 195.2 73 1000 890.5 442.6 50 3000 2627.6 1542.0 59 Summary table 1.0 Dilution Recovery from neat Recovery from neat Recovery from neat Recovery from neat Recovery from neat factor (DF) (%) (%) (%) (%) (%) 1 100 100 100 100 100 2 119 113 95 97 92 4 128 135 93 96 85 8 137 150 90 96 82 16 139 146 92 95 83 32 149 145 94 99 83 Cytokines and mouse/rat fluids were selected according to availability of mouse/rat PerkinElmer AlphaLISA kits, as well as availability and cost of fluids from suppliers. Expected physiological range: Recovery from neat (%) 9 Step 2: 1,000 Dilution factor (DF) r2 neat = 0.9552 4.0 r2 dil 1/2 = 0.9982 r2 neat = 0.9827 4.0 r2 dil 1/2 = 0.9998 Diluent: PBS-0.1%BSA 5 L Standard or animal fluid Incubate 30 minutes Rabbit AF Step 2: Step 3: Evaluate spike and recovery value: • • • • • PBS - 0.1%BSA 5.0 Dilution Step 2: Diluent selection: HiBlock buffer PBS - 0.01%BSA NACL buffer Mouse and rat IL6, TNFα and MCP-1 detection: AlphaLISA® kits available from PerkinElmer: IL6 (mouse) (AL504), TNFα (mouse) (AL505) and CCL2/MCP-1 (mouse/rat) (AL509). Assay buffer is AlphaLISA Immunoassay Buffer (AL000C) for mouse IL6 (mIL6) and mouse TNFα (mTNFα), and NaCl Buffer (AL007C) for mouse MCP-1 (mMCP-1). All calibration curves were done using mouse analyte supplied in the kit. All 3 kits similarly detect mouse and rat cytokines. Fluids pre-treatment and storage: Prior to being used in AlphaLISA assays: FBS was triple filtered at 0.1µm by Wisent; mouse serum was filtered at 0.2µm by Bioreclamation LLC; BALF and AF fluids were centrifuged at 650xg for 15 min at 4˚C to remove cells and cell debris, and supernatants were used in assays; CSF fluids were not pretreated. Storage: Mouse serum and FBS were kept at -20˚C. All other fluids (BALF, CSF, AF) were kept at -80˚C. Example (½ dilution required) Step 1: Linearity of dilutions in 5 diluents for spiked rat AF (3ng/mL mMCP-1) Spiked sample fluid (3ng/mL or 10ng/mL mouse cytokine) Mouse serum Mouse BALF Rat CSF Rat AF Materials and Methods Diluents tested: AlphaLISA HiBlock Buffer: PerkinElmer, cat# AL004C. Foetal bovine serum Premium (FBS): Wisent, cat# 080150. Fluids tested as diluents were supplied as pools of individuals, non-medicated, non-immunized, from Bioreclamation LLC: • Beagle bronchial lavage fluid (BALF): cat# BGL-BROLAV • Beagle cerebrospinal fluid (CSF): cat# BGLCSF • Rabbit amniotic fluid (AF): Strain New Zealand White, cat# RABAMNFL 8 Experimental approach Step 1: Perform linearity experiment with the following diluents for all 3 cytokines: Add 25 L of Streptavidin Alpha Donor beads 2 6 Alpha Technology Assay Principle Concentration (ng/mL) Abstract Concentration (ng/mL) 1 10 Sample fluid Best diluent Mouse serum FBS Mouse BALF PBS-0.1%BSA Rat CSF Beagle CSF Rat AF PBS-0.01%BSA Mouse serum FBS Mouse BALF PBS-0.1%BSA Rat CSF PBS-0.1%BSA Rat AF PBS-0.1%BSA Mouse serum FBS Mouse BALF Beagle BALF Rat CSF PBS-0.1%BSA Rat AF PBS-0.1%BSA Dilution of Physiological Spike range in LDL in sample fluid level sample fluid diluent required in measured giving good (pg/mL) diluent (pg/mL) recovery (pg/mL) 2.1 1/2 33 30 - 10000 2.4 Neat 22 10 - 3000 3.4 Neat ND 10 - 3000 2.9 1/2 ND 10 - 3000 1.6 1/2 55 30 - 3000 1.6 Neat 17 10 - 3000 1.4 Neat ND 3 - 3000 1.4 1/2 5 3 - 3000 5.4 1/2 444 300 - 3000 5.7 Neat 22 10 - 3000 2.9 Neat 501 100 - 3000 2.4 1/2 1151 300 - 3000 Conclusion • Sensitive bead-based immunoassays were developed to detect IL6, TNFα and MCP-1 from rodent serum, BALF, CSF and AF. Good recoveries were obtained from fluids spiked as low as 3 pg/mL. • This study highlights that the appropriate diluent is not easy to predict. In some cases where a perfect match of diluent and fluid is not found, sample dilution is required. • These no wash rapid assays require only 5µL sample volumes. This represents a substantial improvement over ELISA-type assays in terms of speed of execution and sample consumption. • Moreover, results presented demonstrate that measured physiological levels are also in agreement with the literature. PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com