Recombinant Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)

Blocking/Diluting buffer and concentration 5% NFDM/TBST

Paraffin-embedded mouse kidney tissue stained for Polyethylene glycol using purified ab51257 at 1/50 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer) secondary. Counterstained with Hematoxylin. No staining on mouse kidney without PEGylated protein treatment (A), positive staining on mouse kidney with PEGylated protein treatment (B).

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Comparison of sandwich ELISA using RabMAb/RabMAb (ab51257/ab51257) and MAb/RabMAb (Vendor A/ab51257) for capture/detection.

Ab51257/ab51257*: Plate coated with 5 ug/mL of #47; 5 ug/mL of #47 used for detection (*Anti-PEG 47 biotin labeled).

Vendor A/ ab51257*: Plates coated with 100 ug/mL of Vendor A Mouse MAb; 5 ug/mL of #47 used for detection (*Anti-PEG 47 biotin labeled).

ab51257, unpurified,  staining mouse kidney at 1:10 dilution. Left panel: without animal injection with a PEGylated protein. Right panel: with animal injection with a PEGylated protein.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

ab51257, unpurified, staining mouse muscle at 1:10 dilution. Left panel: without animal injection with a PEGylated protein. Right panel: with animal injection with a PEGylated protein.

ab51257, unpurified, staining mouse liver at 1:10 dilution. Left panel: without animal injection with a PEGylated protein. Right panel: with animal injection with a PEGylated protein.

ab51257, unpurified, staining mouse spleen at 1:10 dilution. Left panel: without animal injection with a PEGylated protein. Right panel: with animal injection with a PEGylated protein.

Immunohistochemistry images of anti-PEG (brown) and hematoxylin counterstaining (blue) of tumor (A1–A3 and B1–B3) and muscle tissue (A4 and B4) 30 minutes (A) and 2 h (B) post injection of Spago Pix

Tumor tissue at both time points is shown at three different enlargements where the enlarged areas are indicated by the square boxes. Vessels (V), fibroblasts (Fb) and fat cells (Fc) are indicated with rounded lines. Note that the tumor tissue in panel B1–B3 originates from a mouse injected with a dose of 10 µmol Mn/kg, whereas the other tissues originate from animals administered 20 µmol Mn/kg.

Paraformaldehyde fixed muscle and tumor tissues collected before injection and at 30 minutes and 2–4 h post-dose were paraffin embedded and thereafter cut (4 µm) and positioned on glass tissue slides. Prior to staining, the slides were placed in an antigen-retrieval solution using an automated pre-treatment module (PT-Link; Dako, Glostrup, Denmark). Slides were stained for PEG in an automated immunohistochemistry robot (Autostainer; Dako). PEG is detected with ab51257 at 0.6 μg/ml.

(From Figure 6 Eriksson et al)

Method：Protein A sensor + Antigen (PEG 5K-BSA at 0.3µg/ml)+ antibody (ab51257 at  0.0312, 0.0625, 0.125, 0.25, 0.5, 1 µg/m).

Results:

Sample ID: PEG-5K-BSA

KD (M): 2.41E-09

kon(1/Ms): 2.67E+04

kon Error: 6.81E+02

kdis(1/s): 6.43E-05

kdis Error: 2.92E-06

Full X^2: 0.018444

Full R^2: 0.982059

Comparison of 10 ug/well of activated linear (PEG5K) and branched (PEG40K) PEG using 5 ug/ml of ab51257 in Direct ELISA assay.

Accuracy: By detecting the methoxy group of the PEG molecule itself, ab51257 is useful in measuring the pharmacokinetics of PEG-modified molecules in vivo. Data indicate that ab51257 detects various length Y-chain PEG molecules as well as single chain PEG molecules with equal affinity. ab51257 does not cross react with non-specific targets in blood or serum.

Goat anti-rabbit IgG-AP used for anti-PEG-47 detection; goat anti-mouse IgM-AP used for Vendor A MAb detection.

Fig 1a. Direct ELISA using 1 ug/mL of PEG-GCSF.

Fig 1b. Direct ELISA using 1 ug/mL of PEG-IFN.

Comparison to other anti-PEG: In both direct and sandwich ELISA assays, ab51257 shows greater affinity and accuracy than other anti-PEG antibodies when determining the concentration of PEG or PEG-modified proteins. Results were similar whether detecting PEG itself or PEG-modified targets.

ELISA Graph generated using purified ab51257 at 1μg/mL.

Antigen concentration range:

PEG5000 and PEG20000: 0.0078 - 0.5 μg/mL
ADI-PEG: 0.0156 - 1 μg/mL
PEG-BSA: 0.0039 - 0.25μg/mL
PEG-IFN-α-2b0.0156 - 1 μg/mL

Secondary antibody was an Alkaline Phosphatase-conjugated Goat Anti-Rabbit IgG(H+L) at 1/2500.