For the quantitative measurement of PEGylated molecules in plasma, serum and cell culture media

Transcription

1 ab Polyethylene Glycol (PEG) ELISA Kit Instructions for Use For the 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.

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3 Table of Contents 1. Introduction 3 2. Assay Summary 5 3. Kit Contents 6 4. Storage and Handling 6 5. Additional Materials Required 6 6. Preparation of Reagents 7 7. Assay Procedure 9 8. Data Analysis Troubleshooting 17 2

4 1. Introduction Polyethylene glycol (PEG) is an O-CH 2 -CH 2 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. ab Polyethylene Glycol (PEG) 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 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. 3

5 Requirements Users must PEGylate their interested molecules. For pharmaco kinectic experiments, the PEGylated molecules will be used to construct standard curves. The use of this kit requires the end user to have at least 250ng of PEGylated compound of interest to use as reference standard. The included mpeg-bsa is a reference sample only. 4

6 2. Assay Summary Prepare all reagents, samples and standards Mix 1X PEG-HRP with the standard series, test samples or control Add the mixture to each well Incubate for 45 min at room temp on a shaker Wash 3 times Combine TMB A and TMB B Solutions and add. Cover and incubate at room temperature for 15 minutes Add Stop Solution Read optical density at 450 nm 5

7 3. Kit Contents Item Dry Plate w/ stripwells Reference Standard (PEG-BSA) Quantity 1 x 96 well plate 200 ng PEG conjugated HRP (60X) 150 µl Antigen/Antibody Diluent Buffer (1X) ELISA Washing Buffer (10X) TMB A Substrate Solution (1X) TMB B Substrate Solution (1X) Stop Solution (1X) 20 ml 12 ml 7 ml 7 ml 11 ml 4. Storage and Handling Store all components at 4 C. 5. Additional Materials Required Deionized water PEGylated sample Pipettors and pipette tips of various sizes Rotating shaker Microtiter plate reader 6

8 6. Preparation of Reagents Microtiter Plate: 1. 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. 2. Replace unrequired strips immediately into the bag, seal and store at 4 C. Buffers: 1. Bring components to room temperature before diluting concentrated reagents with deionized water. 2. Store all buffer and reagents at 4 C when not in use. Reference Standard: 1. Reconstitute lyophilized BSA reference with 1.0 ml of distilled water. Leave the reconstituted standard at room temperature for at least 20 min and mix gently. For PEG-BSA, this reconstitution produces a stock solution of 200 ng/ml*. Create at a 6 point standard dilution series using the amounts listed in Table 1. 7

9 2. 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 2000 and 3000 ng/ml of PEGylated sample and dilute down to about 2 ng/ml. See example using a 4-fold dilution of PEGylated mouse IgG in Table 1. (For further data see Section 8) Standard PEG-BSA (ng/ml) PEG-ms-IgG (ng/ml) (stock)* 2500 Table 1. PEG Labeled Dilution Series 8

10 7. Assay Procedure 1. Prepare all reagents, working standards and samples as directed in the previous section. 2. Remove excess microplate strips from plate and return to foil pouch. 3. Mix 25µl of PEG-HRP (diluted with 1X Antigen/Antibody Diluent Buffer to 1X) with 25µl of the standard series (ie: PEG-BSA, PEG-IgG), test samples or control. 4. Add the 50µl mixture to each well. Incubate for 45 min at room temp on a shaker. 5. Aspirate each well and wash with 250µl 1X wash buffer. Repeat 3 times. 6. Combine TMB A and TMB B (1:1)* Add 100 µl of combined substrate solution to each well. * Volume of each TMB substrate needed = 50µl (# of wells +1) 7. Cover to protect from light and incubate at room temperature for 15 minutes. 8. Add 100 µl of stop solution to each well and tap plate to ensure thorough mixing. 9. Determine the optical density at 450 nm using a microplate reader within 30 minutes. 9

11 8. Data Analysis 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 trendline. The curve will generate the equation: y=ax -B and also generate a R 2 value. (see Typical Data below) Using the mean absorbance value for each sample, determinethe corresponding concentration of sample in ng/ml (x) from the equation: x=10^((log A log y) / B). See Fig 2a. A. Typical Data Fig 1. Competition between PEGylated molecules and HRP-mPEG 10

12 Fig 2a. Using the fitted curve (y = 1.11x ), the IgG concentration (x) for each available OD (y) can be calculated as: 10^((log(1.11)-log(y))/0.34) 11

13 Fig 2b. Plot of PEGylated IgG (ng/ml) concentration over time (hours) IgG ng/ml OD

14 Fig 2c. Plot of PEGylated IgG (ng/ml) concentration over time (hours) B. Sensitivity For migg, 25 ng/ml of mpeg-migg competes against mpeg-hrp 50% efficiently. C. 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 migg in our experiments. Rabbit monoclonal Anti-PEG specifically recognizes the methoxy group of mpeg. 13

15 D. Intra-Assay Precision Cell Culture Media Sample n mean (ng/ml) std dev CV (%) Serum Sample n mean (ng/ml) std dev CV (%)

16 E. Inter-Assay Precision Cell Culture Media Sample n mean (ng/ml) std dev CV (%) Serum Sample n mean (ng/ml) std dev CV (%)

17 F. Recovery The recovery of BSA-PEG spiked to Human serum, plasma, and cell culture medium was evaluated. Sample Type Ave % Recovery Range Serum (n=6) % Plasma (n=6) % Cell Culture Media (n=6) % 16

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

19 Problem Cause Solution Large CV Low sensitivity Bubbles in wells All wells not washed equally/thoroughly Incomplete reagent mixing Inconsistent pipetting Inconsistent sample preparation or storage Improper storage of ELISA kit Using incompatible sample type (e.g. Serum vs. cell extract) Ensure no bubbles present prior to reading plate Check that all ports of plate washer are unobstructed/wash wells as recommended Ensure all reagents/master mixes are mixed thoroughly Use calibrated pipettes and ensure accurate pipetting Ensure consistent sample preparation and optimal sample storage conditions (eg. minimize freeze/thaws cycles) Store all reagents as recommended. Please note all reagents may not have identical storage requirements. Detection may be reduced or absent in untested sample types For further technical questions please do not hesitate to contact us by or phone (select contact us on for the phone number for your region). 18

20 UK, EU and ROW Tel: +44 (0) US, Canada and Latin America Tel: ABCAM (22226) China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print.