ELISA/assay

Human Anti-PEG IgG ELISA

DEIASL243

96T

Human Anti-PEG IgG ELISA

PEG

Human Anti-PEG IgG ELISA

iELISA

PEG

Human

Quantitative

serum, plasma

96T

All components should be stored at 4°C. The microtiter plate should be kept in a sealed bag with desiccant. Kits will remain stable for 12 months from the date of purchase provided that the components are stored as described.

PEG

Human

Polyetheylene Glycol, PEG

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

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 human 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-human IgG monoclonal antibody for detection. Serum and 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. 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 × 8-wells). Store at 4°C 2. Anti-IgG HRP Stock (100×). Store at 4°C 3. Anti-PEG Stock (1000 U/mL). Store at 4°C 4. 20× HRP PEG Wash, 50 ml 5. HRP PEG Diluent, 50 ml 6. TMB, 2 × 6 ml 7. Stop Solution, 7 ml

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

In studies at Creative Diagnostics, Inc., we found that anti-PEG IgG levels in human samples ranged from undetectable to 3750 u/ml. Optimal dilutions must be determined empirically. However, we suggest that samples initially be diluted 20-fold. To avoid matrix effects, do not test dilutions less than 20-fold (i.e., 10- fold).

WASH SOLUTION PREPARATION 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. STANDARD PREPARATION 1.The anti-PEG IgG standard is provided as a 1000 U/mL stock. Dilute the stock to 100 u/mL with HRP PEG Diluent (50 μl 1000 u/mL stock + 450 μl HRP PEG Diluent). Stir to homogeneity. 2.Label 7 polypropylene or glass tubes as 50, 25, 12.5, 6.25, 3.13, 1.56 and 0.78 U/mL. 3.Dispense 250 μl of diluent into the tubes. 4.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. 5.Similarly prepare the remaining standards by serial dilution. HRP CONJUGATE PREPARATION Dilute Anti-igG HRP Stock (100×) to 1× with HRP PEG Diluent before use (990 μl HRP PEG Diluent + 10 μl Anti-igG HRP Stock (100×)). Mix well.

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 250 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 μl of diluted HRP conjugate into each well. 7. Incubate on a plate shaker at 250 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 250 rpm/25°C for 20-minutes. 11. Stop the reaction by adding 50 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.All reagents should be allowed to reach room temperature (25°C) before use. 3.The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. 4.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. 5.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. 6.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 human diagnostic applications.

No. Components Size Storage Conditions 1 PEG-BSA coated plate 12 × 8-wells Store at 4°C 2 Anti-IgG HRP Stock (100×) Store at 4°C 3 Anti-PEG Stock 1000 U/mL Store at 4°C 4 20× HRP PEG Wash 50 mL Store at 4°C 5 HRP PEG Diluent 50 mL Store at 4°C 6 TMB 2 × 6 mL Store at 4°C 7 Stop Solution 7 mL Store at 4°C

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.

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

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 human anti-PEG IgG ELISA kit. This kit is for research use only. Under no circumstances should it be used for therapeutic or human diagnostic applications.