ELISA/assay

Monkey anti-PEG IgM ELISA Kit

DEIASL088

96T

Monkey anti-PEG IgM ELISA Kit

PEG

Monkey anti-PEG IgM ELISA Kit

PEG

Quantitative

serum, plasma

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 2-8°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

Monkey

Polyetheylene Glycol; PEG

This assay primarily detects antibodies directed against the polyoxyethylene backbone of PEG.

Attachment of polyethylene glycol (PEG) 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, it has been reported that repeated injections of PEGylated proteins can induce anti-PEG antibodies that increase the rate of clearance and decrease drug efficacy (accelerated blood clearance, or ABC phenomenon). To aid research in this important area, we have developed a monkey anti-PEG IgM 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-monkey IgM monoclonal antibody (LDI clone 2C11-1-5) 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 IgM molecules are thus sandwiched between immobilized PEG and the detection antibody conjugate. The wells are then washed to remove unbound HRP-labeled antibodies. 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 IgM is proportional to the absorbance at 450 nm and is derived from a standard curve.

1. PEG-BSA coated plate (12 x 8-wells) Store at -20°C. 2. Anti-Monkey IgM HRP Stock Store at -20°C. 3. Reference Stock (lyophilized) Store at -20°C. 4. 20x HRP PEG Wash: PEGW50-20, 50 mL 5. HRP PEG Diluent: PEGD50-1, 50 mL 6. TMB: TMB11-1, 11 mL 7. Stop Solution: SS11-1, 11 mL

1. Pipettors and tips 2. Distilled or deionized water 3. Polypropylene or glass tubes 4. Vortex mixer 5. Absorbent paper or paper towels 6. Plate incubator/shaker 7. Plate washer 8. Plate reader capable of measuring absorbance at 450 nm 9. Curve fitting software

Studies at Creative Diagnostics, Inc. indicated that anti-PEG IgM levels were 1756 ± 979 u/mL (mean ± SD, n = 3) prior to immunization. Eight days after immunization with PEG-KLH, levels increased to 6873 ± 2785 u/mL (mean ± SD, n = 3). The fold increase varied from 2.2 to 8.8. Levels may vary with the immunization protocol and the PEG carrier protein used. We suggest that samples initially be diluted 200- fold, but optimal dilutions must be determined empirically. A 200-fold dilution may be achieved by diluting 2.0 μL of serum or plasma with 398 μL of diluent. To avoid matrix effects do not use dilutions less than 50-fold.

WASH SOLUTION PREPARATION: The wash solution is provided as a 20x stock. Prior to use, dilute the contents of the bottle (50 mL) with 950 mL of distilled or deionized water. HRP CONJUGATE PREPARATION: Approximately 5 minutes before needed, dilute the HRP Conjugate stock with diluent (equilibrated to room temperature) as directed on the vial label. STANDARD PREPARATION: 1. The monkey anti-PEG IgM standard is provided as a lyophilized stock. Reconstitute as described on the vial label. 2. Label 5 polypropylene or glass tubes as 100, 50, 25, 12.5, and 6.25 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, and 6.25 u/mL standards by serial dilution.

1. Secure the desired number of coated wells in the holder. 2. Dispense 100 μL 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 1x 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 μL 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 μL 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 μL 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.

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 IgM 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 IgM levels in serum collected ~7 days after immunization with PEG. Serum collected at post-immunization times greater than 7 days may contain high levels of anti-PEG IgG that compete with anti-PEG IgM 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 and/or mixing speeds will likely 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.

1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of and in accordance with the instructions detailed above. 2. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.

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1 N

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