Anti-PEG antibody ELISA (human IgG specific)

Polyethylene glycol (PEG) chains are often used to modify therapeutic biologic agents in order to prolong the circulating half-life of the modified protein. It has been reported that repeat injections of PEGylated proteins can induce anti-PEG antibodies.

Product Specifications

CAS Number

7732-18-5

Gene ID

N/ap

Reactivity

Human

Reaction Volume

15 µL

Target Antigen

Anti-PEG human IgG antibodies

Detection Method

Peroxidase / OD450

Assay Type

ELISA

Assay Principle

Quantification of human IgG antibodies to PEG

Assay Protocol

This immunogenicity assay uses the direct ELISA technique. The supplied 96 well microplate is pre-coated with PEG.

Assay Performance Time

2.5 hours

Sample Type

Serum Plasma

Sample Volume

15ul

Detection Range

62.5 ng/mL -1000 ng/mL

Precision

<10%, <10%

Sensitivity

Anti-PEG antibodies

Limit Of Detection

62.5 ng/mL

Components

Coated microtiter plate, 96 wells QC samples - 6x250ul 10X wash buffer - 50 ml Assay buffer - 50ml 1000X detection reagent - 17ul TMB - 12ml TMB stop solution - 12ml Plate sealers - 3

Shipping Conditions

Blue Ice

Storage Conditions

-20C, 1 year

Shelf Life

12 months

Badge

Hot

Specificity

Anti-PEG antibodies

Method

Direct sandwich ELISA

Preservative

None

Preparation

Prepare only the appropriate amount of required reagent on the day of use. Store all reagents as per instructions stated on the label. 1. Wash Buffer (1X) Preparation: Dilute wash buffer concentrate with ultra-pure water 1/10 before use (for example add 2mL concentrate to 18mL ultra-pure water) . Mix well. 2. Secondary antibody (1X) Preparation: Dilute secondary antibody with assay buffer 1/1000 before use (for examples add 12μl concentrate to 12ml of assay buffer) . Mix well. 3. Detection Reagent (1X) Preparation: Dilute detection reagent with assay buffer 1/1000 before use (for example add 12μl concentrate to 12ml of assay buffer) . Mix well.

Operating Procedure

This assay employs the sandwich enzyme immunoassay technique. Immobilized PEG is coated onto a 96 well microplate. Calibrator and test samples are pipetted into the appropriate wells. Anti-PEG antibodies present in biological matrices is bound by the immobilized PEG. After washing away any unbound substances, enzyme linked anti IgG or IgM antibody is added to the wells. The plate is washed to remove any unbound antibody-enzyme reagent and a substrate solution is added to the wells for color development. The color development is proportional to the amount of anti-PEG antibodies present in test samples. The color development is stopped and the intensity of the color is measured

Results Calculation

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. 2. The concentration of the unknowns can be read directly from this standard curve using the absorbance value for each sample. 3. Any sample undiluted or diluted still 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.