ELISA/assay

Monkey anti-PEG IgG ELISA Kit

DEIASL087

96T

Monkey anti-PEG IgG ELISA Kit

PEG

Monkey anti-PEG IgG 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). In our own studies, we find that a single injection of PEGylated protein, induced high anti-PEG IgG titers (refer to the "Sample Preparation" section.) To aid research in this important area, we have developed a monkey 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-monkey IgG monoclonal antibody (LDI clone 6D11-2) for detection. Serum or plasma samples are diluted and incubated alongside standards in the microtiter wells for 1 hour. The wells are subsequently washed. 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. 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, and 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.

1. PEG-BSA coated plate (12 x 8-wells) Store at -20°C. 2. Anti-Monkey IgG 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

General Note: In studies at Creative Diagnostics, Inc., we found that antiPEG IgG levels were undetectable in serum from naïve monkeys. Twenty-four days after a single immunization with mPEG-KLH, levels increased to 36,406 ± 27,336 u/mL (mean ± SD, n = 3, see figure below). Levels will vary with the immunization protocol and the PEG carrier protein used. Optimal dilutions must be determined empirically. However, we suggest that samples initially be diluted 1000-fold using the following procedure. 1. Dispense 95 μL and 294 μL of diluent into separate polypropylene or glass tubes. 2. Pipette and mix 5 μL of the serum sample into the tube containing 95 μL of diluent. This provides a 20-fold diluted sample. 3. Dilute 6 μL of the 20-fold diluted sample into the tube containing 294 μL of diluent and mix. This provides a 1000-fold diluted sample.

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 IgG standard is provided as a lyophilized stock. Reconstitute the stock as described on the vial label. 2. Label 7 polypropylene or glass tubes as 100, 50, 25, 12.5, 6.25, 3.13 and 1.56 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 remaining 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 log10 of the concentration. 2. Fit the standard curve to a four-parameter logistic regression (4PL) equation (x axis = log10 concentration) and determine the concentration of the samples from the standard curve (remember to derive the antilog). 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 is shown below. This curve is for the purpose of illustration only. A standard curve must be run with each experiment.

1. Please read and instructions thoroughly before using the kit. 2. This kit is designed to measure anti-PEG IgG levels in serum collected>21 days after immunization with PEG. Serum collected at post-immunization times less than 21 days may contain high levels of anti-PEG IgM that compete with anti-PEG IgG 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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