ab138914 – (peg) elisa kit polyethylene glycol pe… · introduction 1. background 2 2. assay...

Version 2 Last Updated 4 December 2014

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.

ab138914 –Polyethylene Glycol (PEG) ELISA Kit

Discover more at www.abcam.com 1

Table of Contents

INTRODUCTION1. BACKGROUND 22. ASSAY SUMMARY 4

GENERAL INFORMATION3. PRECAUTIONS 54. STORAGE AND STABILITY 55. MATERIALS SUPPLIED 56. MATERIALS REQUIRED, NOT SUPPLIED 67. LIMITATIONS 68. TECHNICAL HINTS 7

ASSAY PREPARATION9. REAGENT PREPARATION 810. STANDARD PREPARATIONS 911. PLATE PREPARATION 13

ASSAY PROCEDURE12. ASSAY PROCEDURE 14

DATA ANALYSIS13. CALCULATIONS 1514. TYPICAL DATA 1615. ASSAY SENSITIVITY 2016. ASSAY SPECIFICITY 20

RESOURCES17. TROUBLESHOOTING 2118. NOTES 23


INTRODUCTION

1. BACKGROUND

Abcam’s Polyethylene Glycol (PEG) RabMab® in vitro ELISA (Enzyme-Linked 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 water-soluble, 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.


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


INTRODUCTION

sample will result in decreased or no color development.


GENERAL INFORMATION

3. PRECAUTIONSPlease 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 STABILITYStore 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

Item AmountStorage

Condition(Before

Preparation)Dry Plate w/ stripwells (12 x 8 well RabMab® antibody coated strips) 1 x 96 well plate +2-8°C

Reference Standard (PEG-BSA) 1 x 200 ng + 2-8°C

PEG conjugated HRP (60X) 1 x 150 µL +2-8°C

Antigen/Antibody Diluent Buffer (1X) 1 x 20 mL + 2-8°C

ELISA Washing Buffer (10X) 1 x 12 mL +2-8°C

TMB A Substrate Solution (1X) 1 x 7 mL + 2-8°C

TMB B Substrate Solution (1X) 1 x 7 mL +2-8°C

Stop Solution (1X) 1 x 11 mL + 2-8°C


GENERAL INFORMATION

6. MATERIALS REQUIRED, NOT SUPPLIEDThese 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.


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 or bubbles when mixing or reconstituting 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.


ASSAY PREPARATION

9. REAGENT PREPARATION Equilibrate all reagents and samples to room temperature (18-

25°C) prior to use.

Store all buffers and reagents at 4˚C when not in use. 9.1 1X PEG conjugated HRP

Equilibrate PEG conjugated HRP (60X) to room temperature before diluting to 1X with Antigen/Antibody Diluent Buffer to 1X.

9.2 1X ELISA Washing BufferEquilibrate ELISA washing buffer (10X) to room temperature before diluting to 1X with deionized water.


ASSAY PREPARATION

10.STANDARD PREPARATIONS Prepare serially diluted standards immediately prior to use.

Always prepare a fresh set of standards for every use.

10.1 PEG - BSA Reference Standard Dilution10.1.1 Label seven tubes with BSA reference Standards

#1- 7.10.1.2 Reconstitute lyophilized BSA reference with 1 mL of

distilled water. Leave the reconstituted standard at room temperature for at least 20 minutes and mix gently. For PEG-BSA, this reconstitution produces a stock solution of 200 ng/mL. Transfer 350 μL of this stock solution to tube #1 to create PEG-BSA Standard #1.

10.1.3 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.

10.1.4 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.

10.1.5 Using the table below as a guide to create PEG-BSA Standards #4 through #6.

10.1.6 PEG-BSA Standard #7 contains no protein and is the Blank control.


ASSAY PREPARATION

PEG-BSA

Standard #

Sample to Dilute

Volume to Dilute(µL)

Volume of

Diluent (µL)

StartingConc.

(ng/mL)

Final Conc.

(ng/mL)

1 See Step 10.1.22 Standard #1 225 75 200 1503 Standard #2 133 67 150 1004 Standard #3 100 100 100 505 Standard #4 40 160 50 106 Standard #5 40 160 10 2

7 (Blank) N/A NA 200 0 0


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.

10.2 PEGylated Sample Standard Dilution10.2.1 Label seven tubes with PEG-Mouse-IgG Standards

#1 - 7.10.2.2 Reconstitute / dilute PEG-Mouse-IgG with distilled

water to produce a stock solution of 2,500 ng/mL. Transfer 200 μL of this stock solution to tube #1 to create PEG-Mouse-IgG Standard #1.

10.2.3 Add 150 μL distilled water into tube #2 - 6 and 150 μL distilled water into tube #7.

10.2.4 Prepare PEG-Mouse-IgG Standard #2 by transferring 50 μL from PEG-Mouse-IgG Standard #1 to tube #2. Mix thoroughly and gently.

10.2.5 Prepare PEG-Mouse-IgG Standard #3 by transferring 50 μL from PEG-Mouse-IgG Standard #2 to tube #3. Mix thoroughly and gently.

10.2.6 Using the table below as a guide, repeat for tubes #4 through #6.

10.2.7 Sample Standard #7 contains no protein and is the Blank control.


ASSAY PREPARATION

PEG-Mouse-

IgG Standard

#

Sample to Dilute

Volume to Dilute(µL)

Volume of

Diluent (µL)

StartingConc.

(ng/mL)

Final Conc.

(ng/mL)

1 See Step 10.2.22 Standard #1 50 150 2,500 6253 Standard #2 50 150 625 1574 Standard #3 50 150 157 395 Standard #4 50 150 39 9.86 Standard #5 50 150 9.8 2.4

7 (Blank) N/A N/A 150 0 0


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.


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.12.1 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.

12.2 Add the 50 µL mixture to each well. Incubate for 45 min at room temp on a shaker.

12.3 Aspirate each well and wash with 250 µL 1X Wash Buffer. Repeat 3 times.

12.4 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.

12.6 Add 100 µL of stop solution to each well and tap plate to ensure thorough mixing.

12.7 Determine the optical density at 450 nm using a microplate reader within 30 minutes.


DATA ANALYSIS

13.CALCULATIONSCalculate 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 DATATYPICAL STANDARD CURVE – Data provided for demonstration purposes only. A new standard curve must be generated for each assay performed.


DATA ANALYSIS

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)

PEG-Mouse-IgG ng/mL O.D 450 nm

2,500 0.076625 0.097157 0.25039 0.4559.8 0.5712.4 0.631


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.


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 (ab138914).Competition between PEGylated molecules and HRP-mPEG


DATA ANALYSIS

RECOVERY – (The recovery of BSA-PEG spiked to Human serum, plasma, and cell culture medium was evaluated).

Sample Type Average % Recovery Range (%)

Serum (n=6) 113 90-122Plasma (n=6) 100 85-114Cell Culture Media (n=6) 106 85-117

PRECISION–(Intra-Assay) Cell Culture Media1 2 3

n= 20 20 20Mean (ng/mL) 40.42 20.96 60.29

SD 6.31 3.44 5.12%CV 15.62 16.42 8.49

PRECISION–(Intra-Assay) Serum1 2 3

n= 20 20 20Mean (ng/mL) 51.83 24.02 68.81

SD 10.2 3.4 3.33%CV 19.7 14.3 4.85

PRECISION–(Inter-Assay) Cell Culture Media1 2 3

n= 20 20 20Mean (ng/mL) 41.46 24.84 57.96

SD 3.99 2.55 8.31%CV 9.67 10.28 14.34

PRECISION–(Inter-Assay) Serum1 2 3

n= 20 20 20Mean (ng/mL) 46.67 31.36 61.24

SD 4.14 5.34 11.14%CV 8.87 17.01 18.19


DATA ANALYSIS

15.ASSAY SENSITIVITYFor mouse IgG, 25 ng/mL of mPEG-mouse-IgG competes against mPEG-HRP 50% efficiently.

16.ASSAY SPECIFICITYMonomethoxy 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.


RESOURCES

17.TROUBLESHOOTING

Problem Cause Solution

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

Poor standard curve

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

Low signal

Sample prepared incorrectly

Ensure proper sample preparation/dilution

Wells are insufficiently washed

Wash wells as per protocol recommendations

High background

Contaminated wash buffer

Make fresh wash buffer


RESOURCES

Problem 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

Large CV

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.

Low sensitivity

Using incompatible sample type (e.g. Serum vs. cell extract)

Detection may be reduced or absent in untested sample types


RESOURCES

18.NOTES


RESOURCES

RESOURCES 27

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China and Asia Pacific

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ab138914 – (peg) elisa kit polyethylene glycol pe… · introduction 1. background 2 2. assay...

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