AffinityImmuno

ELISA/assay

Anti PEG antibody ELISA (human IgM specific)

EL-141-PEG-hIGM

1X96 wells

599.99 USD

EL-141-PEG-hIGM

Anti PEG antibody ELISA (human IgM specific)

ELISA

599.99 USD

479.99

USD

1X96 wells

-20C, 1 year

PEG, 5000Da

Human, mouse, rat

250ng/ml-62.5ng/ml

62.5ng/ml

INTRODUCTION Polyethylene glycol (PEG) chains are often used to modify therapeutic biologic agents in order to prolong the circulating half-life of the modified protein by slowing proteolytic degradation. It has been reported that repeat injections of PEGylated proteins can induce anti-PEG antibodies. Anti-PEG antibodies can result in rapid clearance and decreased drug efficacy (accelerated blood clearance, or ABC, phenomenon). PRINCIPLE OF THE ASSAY This immunogenicity assay measures the presence of human IgM in serum or plasma that bind to immobilized PEG using the direct ELISA technique. The supplied stretavidin precoated 96 well microplate is incubated with biotinylated PEG (5KDa). After washing, quality control and test samples are pipetted into the appropriate wells. Anti-PEG antibodies bind the immobilized PEG. After washing, detection antibody (anti human IgM Peroxidase) is added. Any unbound antibody enzyme reagent is removed with a final wash and a substrate solution is added to the wells for color development. Color development is proportional to the amount of anti-PEG IgG.

Coated microtiter plate, 96 wells QC samples - 6x250ul 10X assay/wash buffer - 50ml 500X biotinylated PEG(5KDa) - 50ul 1000X detection reagent - 17ul TMB - 12ml TMB stop solution - 12ml Plate sealers - 3

Indirect ELISA

Peroxidase / OD450

Quantification of human IgM antibodies to PEG

Strip

Serum Plasma

100ul

3.5 hours

PEG

<10%, <10%

PEGylated protains may induce an antibody repsonse altering the normal pharmacokinetic profile of the drug.

This immunogenicity assay uses the indirect ELISA technique. The supplied stretavidin precoated 96 well microplate is incubated with biotinylated PEG (5KDa). After washing, quality control and test samples are pipetted into the appropriate wells. Anti-PEG antibodies bind the immobilized PEG. After washing, detection antibody is added. Any unbound antibody enzyme reagent is removed with a final wash and a substrate solution is added to the wells for color development. Color development is proportional to the amount of anti-PEG antibody.

Prepare only the appropriate amount of required reagent on the day of use. Store all reagents as per instructions stated on the label. 1. Assay/Wash Buffer (1X) Preparation: Dilute assay/wash buffer concentrate with ultra-pure water 1/10 before use (for example add 50mL concentrate to 450mL ultra-pure water). Mix well. 2. Biotinylated PEG (1X) Preparation: Dilute Biotinylated PEG with assay/wash buffer 1/500 before use (for examples add 24μl Biotinylated PEG to 12ml of assay/wash buffer). Mix well. 3. Detection Reagent (1X) Preparation: Dilute detection reagent with assay/wash buffer 1/1000 before use (for example add 12μl concentrate to 12ml of assay/wash buffer). Mix well.

1. Construct a standard curve by plotting the absorbance obtained from each standard against concentration. Use a 4 or 5 parameter curve fit. Alternatively a log-log curve fit may be used. The concentration of the unknowns can be read directly from this standard curve using the absorbance value for each sample. 2. We recommend each lab develop their own statistical cutpoint using methodologies as described by G. Shankar, et al. (2008). (Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products. J. Pharmaceutical and Biomedical Analysis 48:1267– 1281). 3. Any sample reading greater than the highest standard should be diluted appropriately with assay buffer and retested. If the samples have been diluted, the concentration determined from the standard curve must be multiplied by the dilution factor.

This kit is compatible with EDTA-plasma, heparinplasma and serum samples. Samples can be stored at or below -20°C for up to 1 year.

Use QC samples and unknown samples neat. If unknown samples are out of range, then dilute accordingly using supplied assay buffer.