Anti-PEG

Suppliers of anti-PEG antibodies since 1999

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More info:

Dr. Steve Roffler

IBMS

Academia Sinica

sroff@ibms.sinica.edu.tw

(Tel) 886-2-2652-3079

Ms. Mei Lee

Licensing Associate

Academia Sinica

PEG@gate.sinica.edu.tw

(Tel) 886-2-2787-2503

(Fax) 886-2-2651-8049

Links:

- Literature
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Anti-PEG Antibody FAQ

Contents

Anti-PEG antibodies

1. What are anti-PEG antibodies?
2. What is the subclass of the antibodies?
3. What animal are the antibodies from?
4. Are preservatives present?
5. Are carrier proteins present?
6. What buffer are the antibodies shipped in?
7. How should I store the antibodies?
8. How long can the antibodies be stored?
9. Are there any publications regarding these antibodies?
10. Has anyone else used these antibodies?

Antibody specificity

1. What do the antibodies bind to?
2. Can the antibodies bind free PEG?
3. Can the antibodies bind to branched-chain PEG? long PEG?
4. Does the terminal group or linker affect binding?
5. Can the antibodies assay PEGylated compounds in serum samples?
6. Does Tween-20 interfere with the antibodies?

Anti-PEG ELISA

1. Which antibodies should I use for ELISA?
2. What kind of molecules can be assayed by anti-PEG ELISA?
3. What is the detection limit of the assay?
4. Do you have a standard protocol for an anti-PEG ELISA?
5. Can I coat AGP3 and use AGP3 for detection also?
6. Can I use lower concentrations of the antibodies for coating/detection?
7. Can I premix biotin and streptavidin detection reagents?

Antibody quality control

1. What quality control are performed for each lot?
2. Are the antibodies purified?
3. How big is each lot?
4. Is there lot to lot variation?
5. Can you supply enough antibody for our project?

Ordering

1. How do I purchase the antibodies?
2. Can I get a sample?
3. Can I order less than 1 mg antibody?
4. Can we test different lots to find the one that works best for us?
5.  I am a researcher at a university or non-profit organization. How can I get some of your antibodies?
6. Who do I contact with ordering questions?

Shipping

1. How do you ship the antibodies?
2. When will we get our order?
3. Can we use our FedEx account?
4. Who can I contact to open a World Courier account?
5. Who can I contact to open a Midnite Express account?
6. Is there sufficient dry ice in my shipment?

Problems and Their Solutions

1. Where can I get my technical question answered?
2. Who do I contact about ordering antibody?
3. Where can I get help about shipping my order?
4. My background is too high.
5. The signal is too low.
6. My assay is not working as expected. Who can help me?
7. I can't afford the antibodies. Can you give me some for free?

The FAQ

Anti-PEG Antibodies

1. Q: What are anti-PEG antibodies?

    

   A:  Our anti-PEG antibodies are monoclonal antibodies that bind to the repeating subunits present in the polyethylene glycol polymer. These antibodies can be employed to detect and assay PEGylated molecules, including PEGylated proteins, particles and liposomes.

    

    

2. Q: What is the subclass of the antibodies?

    

   A:  AGP3 and AGP4 are IgM antibodies. Appropriate anti-IgM secondary antibodies should be used to detect AGP3 and AGP4.

    

    

3. Q: What animal are the antibodies from?

    

   A:   The antibodies are derived from mice. You should use anti-mouse secondary antibodies for detection.

    

    

4. Q: Are preservatives present?

    

   A:   We add 0.02% sodium azide to the antibodies. We can prepare antibodies without preservatives as a special order (no extra charge). All antibodies are also filter sterilized.

    

    

5. Q: Are carrier proteins present?

    

   A:   No.

    

    

6. Q: What buffer are the antibodies shipped in?

    

   A:   The antibodies are shipped in PBS (0.14 M  NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄). We add 50% glycerol to biotinylated antibodies or to antibodies if they are not shipped on dry ice.

    

    

7. Q: How should I store the antibodies?

    

   A:   We recommend that you aliquot the antibodies into working amounts and freeze them at -80^oC (or -20^oC) for long-term storage. The antibodies are also stable for at least one month at 4⁰C, as long as they are not contaminated with microbes. You can also add 50% glycerol to the antibodies and keep them for at least 1 year at -20^oC. Do not dilute the antibodies before storing them! Do not repeatedly freeze-thaw the antibodies.

    

    

8. Q: How long can the antibodies by stored?

    

   A:   They should be stable for a minimum of 3 years when stored undiluted at -80^oC.

    

    

9. Q: Are there any publications regarding these antibodies?

    

   A:   Yes, several papers concerning these anti-PEG antibodies have been published. Pdf files of some papers are available.

    

    

10. Q: Has anyone else used these antibodies?

     

    A:   More than 80 pharmaceutical and biotechnology companies have purchased our anti-PEG antibodies

     

     

Antibody specificity

1. Q: What do the antibodies bind to?

    

   A The antibodies bind to the repeating subunits present in the polyethylene glycol polymer. The minimal binding domain appears to correspond to PEG₁₀₀₀ (PEG with a molecular weight of 1000 Da).

    

    

2. Q: Can the antibodies bind free (unconjugated) PEG?

    

   A:   Yes and No.

    

   Yes: The antibodies can bind to PEG as long as it is "immobilized". So, for example, amino-terminal PEG can stick to ELISA plates. The antibodies bind very strongly to such PEG-coated microtiter plates. Likewise, the antibodies bind to PEG in immunohistochemical sections and to PEG on nitrocellolose paper. The antibodies bind best to PEG that is physically attached to proteins, liposomes, cells and nanoparticles.

    

   No: Free PEG chains in solution are bound with low affinity by the antibodies. Thus, detection of free PEG molecules by ELISA is not very sensitive. This means that unconjugated PEG will not interfere with assays.

    

    If you need to measure the concentration of unconjugated PEG, you can use our newly developed cell-based assay system.

    

    

3. Q: Can the antibodies bind to branched chain PEG? Long PEG?

    

   A:   Yes, they can! In fact, since the antibodies bind to the PEG backbone, assay sensitivity increases as more PEG chains are present or as the length of the PEG chain increases.

    

    

4. Q: Does the terminal group or linker affect binding?

    

   A:  No. Since our antibodies bind to the PEG backbone, the terminal group and linker chemistry don't influence binding. This is in contrast to "anti-PEG" antibodies that bind the terminal methoxy group present in some PEG molecules.

    

    

5. Q: Can the antibodies assay PEGylated compounds in serum samples?

    

   A: Yes! Serum does not appear to adversely affect the binding of our anti-PEG antibodies to PEGylated compounds.

    

6. Q: Does Tween-20 interfere with the antibodies?

    

   A: No. The epitope recognized by the anti-PEG antibodies is much larger than the PEG-link regions of Tween-20. This detergent can safely be used in ELISA assays. CHAPS can also be used instead of Tween-20.

    

    

Anti-PEG ELISA

1. Q: Which antibodies should I use for ELISA?

    

   A:   To date, we suggest using AGP4 as a capture antibody at 0.25 ug/well (96-well microtiter plates). AGP3-biotin, 3.3-biotin or AGP4-biotin can be used as the detection antibodies. 3.3-biotin usually works best. Streptavidin-HRP (we use SA-HRP from Jackson Laboratories) can then be used to visualize binding. The detection antibodies have slightly different binding abilities depending on the nature of the PEGyated compound. We have published a paper (anti-PEG ELISA) comparing differnet antibodies for sandwich ELISA.

    

    

2. Q: What kind of molecules can be assayed by anti-PEG ELISA?

    

   A:   The types of molecules that we or others have assayed include many different PEG-modified proteins, PEG-modified liposomes and PEG-modified nanodots.

    

    

3. Q: What is the detection limit of the assay?

    

   A:   The detection limit depends strongly on what you are assaying, Typically, a sandwhich assay can detect ~ 1 pM PEGylated q-dots, 1 ng/ml lipodox and 1 ng/ml Pegasys.

    

    

4. Q: Do you have a standard protocol for an anti-PEG ELISA?

    

   A:   Yes. We have specific assays depending on the antibody combinations you wish to use:

    

                

   

   AGP3 as capture antibody  ----   AGP3-biotin or AGP4-biotin detection

    

    

   

   AGP3 as capture antibody  ------ 3.3-biotin detection

    

    

    

   

   AGP4 as capture antibody  ----   AGP3-biotin or AGP4-biotin detection

    

    

   

   AGP4 as capture antibody  ------ 3.3-biotin detection

    

    

    

5. Q: Can I coat AGP3 and use AGP3 for detection also?

    

   A:   No. When you add secondary detection antibody (anti-IgM or anti-Ig conjugate), they will bind AGP3 coated on the plates, giving a high constant background.

    

   However, you can coat AGP3 or AGP4 and then use AGP3-biotin, AGP4-biotin or 3.3-biotin in combination with streptavidin-HRP for detection. This is the recommended assay format for all PEGylated compounds.

    

   You could also coat E11, then detect with AGP3 or AGP4 followed by IgM-specific-HRP conjugate. Likewise, you can coat AGP3 or AGP4 and then detect with E11 followed by IgG-specific-HRP conjugate. However, background binding is usually higher and assay sensitivity is typically not as good as compared to using biotin-labeled antibodies for detection.

    

    

6. Q: Can I use lower concentrations of the antibodies for coating/detection?

    

   A:   Yes, but assay sensitivity will decrease when AGP3 is used as the capture antibody. However, the amount of AGP4 can be reduced to 0.25 mg/well with little impact on assay sensitivity.

    

    

7. Q: Can I premix biotin and streptavidin detection reagents?

    

   A:   Yes!  Premixing AGP3-biotin or AGP4-biotin with streptavidin-HRP before adding them to the ELISA plate is highly recommended. Assay sensitivity is usually increased by premixing (see data below). Assay time is also reduced.

    

   Premixing is NOT recommended when using 3.3-biotin as the detection reagent.

    

    

   

   AGP3-biotin detection of PEGasys

   AGP3 (1 mg/well) capture antibody

   Compare pre-mixed 5 mg/ml AGP3-biotin and 1 mg/ml       streptavidin-HRP versus traditional individual addition method

    

   

   AGP3-biotin detection of Lipo-dox

   AGP3 (1 mg/well) capture antibody

   Compare pre-mixed 5 mg/ml AGP3-biotin and 1 mg/ml streptavidin-HRP versus traditional individual addition method

    

   

   AGP3-biotin detection of Q-dots

   AGP3 (1 mg/well) capture antibody

   Compare pre-mixed 5 mg/ml AGP3-biotin and 1 mg/ml streptavidin-HRP versus traditional individual addition method

    

   

   AGP4-biotin detection of PEGasys

   AGP3 (1 mg/well) capture antibody

   Compare pre-mixed 5 mg/ml AGP3-biotin and 1 mg/ml streptavidin-HRP versus traditional individual addition method

    

   

   AGP4-biotin detection of Lipo-dox

   AGP3 (1 mg/well) capture antibody

   Compare pre-mixed 5 mg/ml AGP3-biotin and 1 mg/ml streptavidin-HRP versus traditional individual addition method

    

   

   AGP4-biotin detection of Q-dots

   AGP3 (1 mg/well) capture antibody

   Compare pre-mixed 5 mg/ml AGP3-biotin and 1 mg/ml streptavidin-HRP versus traditional individual addition method