Anti-PEG antibody ELISA (human IgG specific)

Catalogue Number: EL-141-PEG-hIGG

Size: 1X96 wells

Antigen: Anti-PEG human IgG antibodies

Storage: -20°C, 1 year

Reactivity: Human

Detection Range: 62.5 ng/mL -1000 ng/mL

Detection Minimum: 62.5 ng/mL

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

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

Method Type: Direct sandwich ELISA

Detection Method: Peroxidase / OD450

Principle: Quantification of human IgG antibodies to PEG

Plate: Strip

Sample Type: Serum Plasma

Sample Volume: 15ul

Assay Time: 2.5 hours

Specificity: Anti-PEG antibodies

Assay Precision: <10%, <10%

Preservative: none

Description: 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.

Gene ID: N/ap

Assay 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

Reagent 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.

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.

Sample Collection: 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.

Sample Preparation: Dilute test samples 1/5 with assay buffer before use (for example add 50μl of test sample to 200μl assay buffer). Mix well.