ELISA/assay

Mouse Anti-PEG IgG ELISA Kit

DEIA6159

96T

Mouse Anti-PEG IgG ELISA Kit

PEG

Mouse Anti-PEG IgG ELISA Kit

Indirect-ELISA

PEG

Mouse

Quantitative and qualitative

Serum

96T

The reference stock, HRP conjugate and the PEG-BSA coated plate should be stored at -20°C. All remaining kit components should be stored at 4 °C. The microtiter plate should be kept in a sealed bag with desiccant. Kits Will remain stable for six months from the date of purchase provided that the components are stored as described.

PEG

Mouse

Polyetheylene Glycol ELISA Kits; PEG

This kit is for research use only. Under no circumstances should it be used for therapeutic or diagnostic applications.

The attachment of polyethylene glycol chains to therapeutic biologic agents, a process referred to as PEGylation, prolongs the circulating half-life of the modified protein by slowing proteolytic degradation and by masking it from the immune system. However, repeated injections of PEGylated proteins can induce anti-PEG antibodies that increase the rate of clearance and thereby decrease efficacy (accelerated blood clearance, or ABC phenomenon). To aid research, we have developed a mouse anti-PEG IgG ELISA kit.

The assay uses immobilized mono mPEGylated BSA (20 kDa PEG chain) as the capture antigen (coated on microtiter wells) and horseradish peroxidase (HRP) conjugated anti-mouse IgG antibodies for detection. Serum or plasma samples are diluted and incubated alongside standards in the microtiter wells for 1 hour. The wells are subsequently washed, and HRP conjugate is added and incubated for 45 minutes. Anti-PEG IgG molecules are thus sandwiched between immobilized PEG and the detection antibody conjugate. The wells are then washed to remove unbound HRP-labeled antibodies, and TMB Reagent is added and incubated for 20 minutes at room temperature. This results in the development of a blue color. Color development is stopped by the addition of Stop Solution, changing the color to yellow. Optical density is measured spectrophotometrically at 450 nm. The concentration of anti-PEG IgG is proportional to the absorbance at 450 nm and is derived from a standard curve. This assay primarily detects antibodies directed against the polyoxyethylene backbone of PEG. Studies at CD in mice and rabbits have demonstrated that most anti-PEG antibodies induced by immunization with PEGylated proteins are directed against the PEG backbone.

1. PEG-BSA coated plate(12 x 8-wells), Store at -20°C 2. Anti-Mouse IgG HRP Stock(lyophilized), Store at -20°C 3. Reference Stock 1 (lyophilized), Store at -20°C 4. 20× HRP PEG Wash: 50 ml 5. HRP PEG Diluent: 50 ml 6. TMB: 11 ml 7. Stop Solution: 11 ml 1 Mouse anti-PEG lgG levels are measured in nominal units and are calibrated using pooled ant-PEG mouse serum prepared at CD.

1. Materials required but not provided: 2. Pipettors and tips 3. Distilled or deionized water 4. Polypropylene or glass tubes 5. Vortex mixer 6. Absorbent paper or paper towels 7. Plate incubator/shaker 8. Plate washer 9. Plate reader capable of measuring at 450 nm. 10. Curve fitting software

Studies at CD indicated that anti-PEG IgG levels were undetectable in serum from control mice. However, in serum from PEG-KLH immunized mice, levels of 12,900 to 133,100 u/ml (71056 ± 61394, mean ± SD, n = 8) were found twenty-five days after immunization. Levels will vary with the immunization protocol and the PEG carrier protein used. We suggest that samples initially be diluted 2000-fold, but optimal dilutions must be determined empirically. A 2000-fold dilution may be achieved as follows: 1.Dispense 98 μl and 341.25 μl of diluent into separate tubes 2. Pipette and mix 2 μl of the serum/plasma sample into the tube containing 98 μl of diluent. This provides a 50-fold diluted sample. 3. Mix 8.75 μl of the 50-fold diluted sample with the 341.25 μl of diluent in the second tube. This provides a 2000-fold dilution.

WASH SOLUTION The wash solution is provided as a 20× stock. Prior to use, dilute the contents of the bottle (50 ml) with 950 ml of distilled or deionized water DILUENT The diluent is formulated for measurement PEG antibodies, It is supplied ready to use. DO NOT substitute other buffers. STANDARDS 1. The mouse anti-PEG lgG standard is provided as a lyophilized stock. Reconstitute the stock as described on the vial label. 2 Label 6 polypropylene or glass tubes as 100, 50, 25, 12.5, 6.25 and 3.13 u/ml. 3. In the tube labeled 100 u/ml prepare the 100 u/ml standard as detailed on the stock vial label. 4. Dispense 250 μl of diluent into the remaining tubes 5. Prepare a 50 u/ml standard by diluting and mixing 250 μl of the 100 u/ml standard with 250 μl of diluent in the tube labeled 50 u/ml. 6.Similarly prepare the 25, 12.5, 6.25 and 3.13 u/ml standards by serial dilution. HRP CONJUGATE Approximately 15 minutes before needed, reconstitute the lyophilized HRP conjugate by adding 200 μl of diluent and mix gently. Dilute as described on the vial label to give the working conjugate solution. The reconstituted conjugate stock must be stored at -20°C in a sealed vial if future use is intended.

1. Secure the desired number of coated wells in the holder. 2. Dispense 100 ul of standards and diluted samples into the wells (we recommend testing in duplicate) 3. Incubate on a plate shaker at 150 rpm/25°C for 1 hour. 4. Aspirate the contents of the microtiter wells and wash the wells five times with 1× wash solution using a plate washer (400 μl/well). 5. Strike the wells sharply onto absorbent paper to remove all residual wash solution. 6. Add 100 ul of diluted HRP conjugate into each well. 7. Incubate on a plate shaker at 150 rpm/25°C for 45-minutes. 8. Wash as detailed above 9. Dispense 100 ul of TMB into each well. 10. Incubate on a plate shaker at 150 rpm/25°C for 20-minutes. 11. Stop the reaction by adding 100 ul of stop solution to each well. 12. Gently mix. It is important to make sure that all the blue color changes to yellow. 13. Read the optical density at 450 nm with a microtiter plate reader within five minutes 2 . 2 On some plate readers the A450 value of the high standard may be out of range. If that occurs, absorbance values for all wells may be read at 405 nm instead. Absorbance values will be lower, but this does not affect results.

1. Using curve fitting software, construct a standard curve by plotting absorbance values of the standards versus concentration. 2. Fit the standard curve to a second order polynomial model and determine the concentration of the samples from the standard curve. 3. Multiply the derived concentration by the dilution factor to determine the actual concentration in the samples. 4. If the A450 values of samples fall outside the standard curve, samples should be diluted appropriately and re-tested.

A typical standard curve with optical density readings at 450nm on the Y-axis against anti-PEG IgG concentrations on the X-axis is shown below. This curve is for the purpose of illustration only and should not be used to calculate unknowns.

1. Please read and instructions thoroughly before using the kit. 2. This kit is designed to measure anti-PEG lgG levels in serum collected >14 days after immunization with PEG. Serum collected at post-immunization times less than 14 days may contain elevated levels of anti-PEG lgM that compete with anti-PEG lgG for the immobilized PEG thereby causing interference 3. All reagents should be allowed to reach room temperature (25°C) before use. 4. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. 5. Use only the wash solution and dilution buffer provided with the kit. PEG and PEGylated compounds are found in many buffers conventionally used in ELISA's and cannot be used with this kit 6. Kits are validated using plate shakers set at 150 rpm and 25°C. Performance of the assay at lower temperatures andlor mixing speeds will result in lower absorbance values 7. Optimal results are achieved if, at each step, reagents are pipetted into the wells of the microtiter plate within 5 minutes.

This kit is for research use only. Under no circumstances should it be used for therapeutic or diagnostic applications.

No. Components Size Storage Conditions 1 PEG-BSA coated plate 12 × 8-wells Store at -20°C 2 Anti-Mouse IgG HRP Stock lyophilized Store at -20°C 3 Reference Stock lyophilized Store at -20°C 4 20× HRP PEG Wash 50 ml 2-8°C 5 HRP PEG Diluent 50 ml 2-8°C 6 TMB 11 ml 2-8°C 7 Stop Solution 11 ml 2-8°C

A typical standard curve with optical density readings at 450nm on the Y-axis against anti-PEG IgG concentrations on the X-axis is shown below. This curve is for the purpose of illustration only and should not be used to calculate unknowns.

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet

See the individual product datasheet