ab Polyethylene Glycol (PEG) ELISA Kit

Transcription

1 ab Polyethylene Glycol (PEG) ELISA Kit Instructions for Use For the competitive quantitative measurement of PEGylated molecules in plasma, serum and cell culture media. This product is for research use only and is not intended for diagnostic use. Version 2 Last Updated 4 December 14

2 Table of Contents INTRODUCTION 1. BACKGROUND ASSAY SUMMARY GENERAL INFORMATION 3. PRECAUTIONS 5 4. STORAGE AND STABILITY 5 5. MATERIALS SUPPLIED 5 6. MATERIALS REQUIRED, NOT SUPPLIED 6 7. LIMITATIONS 6 8. TECHNICAL HINTS 7 ASSAY PREPARATION 9. REAGENT PREPARATION STANDARD PREPARATIONS PLATE PREPARATION 13 ASSAY PROCEDURE 12. ASSAY PROCEDURE 14 DATA ANALYSIS 13. CALCULATIONS TYPICAL DATA ASSAY SENSITIVITY 16. ASSAY SPECIFICITY RESOURCES 17. TROUBLESHOOTING NOTES 1

3 INTRODUCTION 1. BACKGROUND Abcam s Polyethylene Glycol (PEG) RabMab in vitro ELISA (EnzymeLinked Immunosorbent Assay) kit is designed for accurate competitive quantitative measurement of PEGylated molecules in plasma, serum and cell culture media. Abcam s Polyethylene Glycol (PEG) RabMab ELISA Kit operates on the basis of competition between enzyme HRP conjugated PEG and PEG labeled molecules for a limited number of binding sites on the surface of 96-wells coated with anti-peg RabMab antibody. The extent of color development resulting from interaction between HRP and the substrate TMB is inversely proportional to the amount of PEGylated molecules in the sample. For example, the absence of PEGylated molecules in the sample will result in a bright blue color, whereas the presence of PEGylated molecules will result in decreased or no color development. Polyethylene glycol (PEG) is an O-CH2-CH2 polymer, which is watersoluble, nontoxic, nonantigenic, and biocompatible. Covalent conjugation of PEG to therapeutic proteins increases the in vivo stability by protecting the protein from degradation, masking its immunogenic sites and reducing clearance. Typically, PEGylation uses nonspecific reactions with nucleophilic residues and produces mixtures of PEGylated positional isomers. Qualitative and quantitative analysis of PEGylated molecules is important for both drug development and clinical application. This kit is developed to determine levels of PEGylated molecules in samples such as serum, plasma or cell culture medium via ELISA. Users must PEGylate their interested molecules. For pharmaco kinetic experiments, the PEGylated molecules will be used to construct standard curves. 2

4 INTRODUCTION The use of this kit requires the end user to have at least 250 ng of PEGylated compound of interest to use as reference standard. The included mpeg-bsa is a reference sample only. 2. ASSAY SUMMARY Remove appropriate number of RabMab antibody coated well strips. Equilibrate all reagents to room temperature. Prepare all the reagents, samples, and standards as instructed. Mix 1X PEG-HRP with the standard series, test samples or controls. Add to appropriate wells. Incubate at room temperature. Aspirate and wash each well. Add substrate solutions. When color develops add the Stop Solution. Immediately begin recording the color development. The presence of PEGylated molecules in the 3

5 INTRODUCTION sample will result in decreased or no color development. 4

6 GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 4. STORAGE AND STABILITY Store kit at +2-8ºC immediately upon receipt. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in the Reagent Preparation section. 5. MATERIALS SUPPLIED Amount Storage Condition (Before Preparation) 1 x 96 well plate +2-8 C 1 x 0 ng C PEG conjugated HRP (60X) 1 x 150 µl +2-8 C Antigen/Antibody Diluent Buffer (1X) 1 x ml C ELISA Washing Buffer (10X) 1 x 12 ml +2-8 C TMB A Substrate Solution (1X) 1 x 7 ml C TMB B Substrate Solution (1X) 1 x 7 ml +2-8 C Stop Solution (1X) 1 x 11 ml C Item Dry Plate w/ stripwells (12 x 8 well RabMab antibody coated strips) Reference Standard (PEG-BSA) 5

7 GENERAL INFORMATION 6. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully utilize this assay: Deionized water PEGylated sample of interest (at least 250 ng to use as a standard) Pipettors and pipette tips of various sizes Rotating shaker Microtiter plate reader 7. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic procedures. Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. 6

8 GENERAL INFORMATION 8. TECHNICAL HINTS Samples generating values higher than the highest standard should be further diluted in the appropriate sample dilution buffers. Avoid foaming components. Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions. Ensure plates are properly sealed or covered during incubation steps. Complete removal of all solutions and buffers during wash steps. This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. or bubbles when mixing or reconstituting 7

9 ASSAY PREPARATION 9. REAGENT PREPARATION Equilibrate all reagents and samples to room temperature (1825 C) prior to use. Store all buffers and reagents at 4 C when not in use X PEG conjugated HRP Equilibrate PEG conjugated HRP (60X) to room temperature before diluting to 1X with Antigen/Antibody Diluent Buffer to 1X X ELISA Washing Buffer Equilibrate ELISA washing buffer (10X) to room temperature before diluting to 1X with deionized water. 8

10 ASSAY PREPARATION 10. STANDARD PREPARATIONS Prepare serially diluted standards immediately prior to use. Always prepare a fresh set of standards for every use PEG - BSA Reference Standard Dilution Label seven tubes with BSA reference Standards # Reconstitute lyophilized BSA reference with 1 ml of distilled water. Leave the reconstituted standard at room temperature for at least minutes and mix gently. For PEG-BSA, this reconstitution produces a stock solution of 0 ng/ml. Transfer 350 μl of this stock solution to tube #1 to create PEG-BSA Standard # Prepare PEG-BSA Standard #2 by adding 75 μl distilled water into tube #2 then transferring 225 μl from PEG-BSA Standard #1 to tube #2. Mix thoroughly and gently Prepare PEG-BSA Standard #3 by adding 67 μl distilled water into tube #3 then transferring 133 μl from PEG-BSA Standard #2 to tube #3. Mix thoroughly and gently Using the table below as a guide to create PEG-BSA Standards #4 through # PEG-BSA Standard #7 contains no protein and is the Blank control. 9

11 ASSAY PREPARATION PEGBSA Standard # (Blank) Sample to Dilute Standard #1 Standard #2 Standard #3 Standard #4 Standard #5 N/A Volume of Diluent (µl) See Step NA 0 Volume to Dilute (µl) Starting Conc. (ng/ml) Final Conc. (ng/ml)

12 ASSAY PREPARATION For the PEGylated sample of interest, make a series dilution using either a 2, 3 or 4-fold dilution depending on number of data points desired. Start with between 2,000 and 3,000 ng/ml of PEGylated sample and dilute down to about 2 ng/ml. Example below with PEG-Mouse-IgG as the PEGylated sample of interest, using a 4-fold dilution from a stock solution at 2,500 ng/ml PEGylated Sample Standard Dilution Label seven tubes with PEG-Mouse-IgG Standards # Reconstitute / dilute PEG-Mouse-IgG with distilled water to produce a stock solution of 2,500 ng/ml. Transfer 0 μl of this stock solution to tube #1 to create PEG-Mouse-IgG Standard # Add 150 μl distilled water into tube #2-6 and 150 μl distilled water into tube # Prepare PEG-Mouse-IgG Standard #2 by transferring 50 μl from PEG-Mouse-IgG Standard #1 to tube #2. Mix thoroughly and gently Prepare PEG-Mouse-IgG Standard #3 by transferring 50 μl from PEG-Mouse-IgG Standard #2 to tube #3. Mix thoroughly and gently Using the table below as a guide, repeat for tubes #4 through # Sample Standard #7 contains no protein and is the Blank control. 11

13 ASSAY PREPARATION PEGMouseIgG Standard # (Blank) Sample to Dilute Standard #1 Standard #2 Standard #3 Standard #4 Standard #5 N/A Volume to Dilute (µl) Volume of Diluent (µl) See Step N/A 150 Starting Conc. (ng/ml) Final Conc. (ng/ml) 2,

14 ASSAY PREPARATION 11. PLATE PREPARATION The 96 well plate strips included with this kit are supplied ready to use. It is not necessary to rinse the plate prior to adding reagents. For each assay performed, a minimum of 2 wells must be used as blanks, omitting primary antibody from well additions. For statistical reasons, we recommend each sample should be assayed with a minimum of two replicates (duplicates). Well effects have not been observed with this assay. Bring stripped microtiter plate to room temperature. Keep appropriate numbers of strips for an experiment and remove extra strips from microtiter plate by evenly pushing the bottoms of the microwell strips. Replace unrequired strips immediately into the bag, seal and store at 4 C. 13

15 ASSAY PROCEDURE 12. ASSAY PROCEDURE Equilibrate all materials and prepared reagents to room temperature prior to use. Prepare all reagents, working standards and samples as directed. It is recommended to assay all standards, controls and samples in duplicate Mix 25 µl of PEG-HRP (diluted with 1X Antigen/Antibody Diluent Buffer to 1X) with 25 µl of the standard series (i.e. PEG-BSA, PEG-IgG), test samples or control Add the 50 µl mixture to each well. Incubate for 45 min at room temp on a shaker Aspirate each well and wash with 250 µl 1X Wash Buffer. Repeat 3 times Combine TMB A and TMB B (1:1) Add 100 µl of combined substrate solution to each well. Note: Volume of each TMB substrate needed = 50 μl x (# of wells +1) 12.5 Cover to protect from light and incubate at room temperature for 15 minutes Add 100 µl of stop solution to each well and tap plate to ensure thorough mixing Determine the optical density at 450 nm using a microplate reader within 30 minutes. 14

16 DATA ANALYSIS 13. CALCULATIONS Calculate the average absorbance values (A450) for each set of standards (or references) and samples. Construct a standard curve by setting concentration of each point in the standard serial dilution in ng/ml along the X axis and the mean absorbance obtained for each standard point as Y axis. Create a curve for the plotted standard dilution series using a power trend line. The curve will generate the equation: y=ax-b and also generate a R2 value. Using the mean absorbance value for each sample, determine the corresponding concentration of sample in ng/ml (x) from the equation: x=10^((log A log y) / B). 14. TYPICAL DATA TYPICAL STANDARD CURVE Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed. 15

17 DATA ANALYSIS PEG-MouseIgG ng/ml 2,500 O.D 450 nm Figure 1. Pharmacokinetic data with PEG ELISA Kit (ab138914). Using the fitted curve (y = 1.11x-0.34), the IgG concentration (x) for each available OD (y) can be calculated as: 10^((log(1.11)-log(y))/0.34) 16

18 DATA ANALYSIS Figure 2. Pharmacokinetic data with PEG ELISA Kit (ab138914). Plot of PEGylated IgG [ng/ml] concentration over time (h). Using four different rat models, also tested negative controls. Figure 3. Pharmacokinetic data with PEG ELISA Kit (ab138914). Plot of PEGylated IgG (ng/ml) concentration over time (hours) in a rat model. Compared against negative control. 17

19 DATA ANALYSIS Figure 4. Sample data with PEG ELISA Kit (ab138914). Plot of migg-peg concentration [ng/ml] detection over time (h). Figure 5. Sample data with PEG ELISA Kit Competition between PEGylated molecules and HRP-mPEG (ab138914). 18

20 DATA ANALYSIS RECOVERY (The recovery of BSA-PEG spiked to Human serum, plasma, and cell culture medium was evaluated). Serum (n=6) Average % Recovery 113 Plasma (n=6) Cell Culture Media (n=6) Sample Type Range (%) PRECISION (Intra-Assay) Cell Culture Media n= Mean (ng/ml) SD %CV PRECISION (Intra-Assay) Serum n= Mean (ng/ml) SD %CV PRECISION (Inter-Assay) Cell Culture Media n= Mean (ng/ml) SD %CV PRECISION (Inter-Assay) Serum n= Mean (ng/ml) SD %CV

21 DATA ANALYSIS 15. ASSAY SENSITIVITY For mouse IgG, 25 ng/ml of mpeg-mouse-igg competes against mpeg-hrp 50% efficiently. 16. ASSAY SPECIFICITY Monomethoxy PEG (mpeg), with the molecular weight about 5 kda, is used to immunize rabbits to generate anti-peg antibody and modify BSA and mouse-igg in our experiments. Rabbit monoclonal RabMab Anti-PEG specifically recognizes the methoxy group of mpeg.

22 RESOURCES 17. TROUBLESHOOTING Problem Cause Solution Poor standard curve Improper standard dilution Confirm dilutions made correctly Standard improperly reconstituted (if applicable) Briefly spin vial before opening; thoroughly resuspend powder (if applicable) Standard degraded Store sample as recommended Curve doesn't fit scale Try plotting using different scale Incubation time too short Try overnight incubation at 4 C Target present below detection limits of assay Decrease dilution factor; concentrate samples Precipitate can form in wells upon substrate addition when concentration of target is too high Increase dilution factor of sample Using incompatible sample type (e.g. serum vs. cell extract) Detection may be reduced or absent in untested sample types Sample prepared incorrectly Ensure proper sample preparation/dilution Wells are insufficiently washed Wash wells as per protocol recommendations Contaminated wash buffer Make fresh wash buffer Low signal High background 21

23 RESOURCES Problem Large CV Low sensitivity Cause Solution Waiting too long to read plate after adding STOP solution Read plate immediately after adding STOP solution Bubbles in wells Ensure no bubbles present prior to reading plate All wells not washed equally/thoroughly Check that all ports of plate washer are unobstructed/wash wells as recommended Incomplete reagent mixing Ensure all reagents/master mixes are mixed thoroughly Inconsistent pipetting Use calibrated pipettes and ensure accurate pipetting Inconsistent sample preparation or storage Ensure consistent sample preparation and optimal sample storage conditions (e.g. minimize freeze/thaws cycles) Improper storage of ELISA kit Store all reagents as recommended. Please note all reagents may not have identical storage requirements. Using incompatible sample type (e.g. Serum vs. cell extract) Detection may be reduced or absent in untested sample types 22

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