Overview

  • Product name
    Anti-Interferon alpha 2b antibody [9D3]
    See all Interferon alpha 2b primary antibodies
  • Description
    Mouse monoclonal [9D3] to Interferon alpha 2b
  • Host species
    Mouse
  • Tested applications
    Suitable for: Sandwich ELISA, Inhibition Assay, WB, Indirect ELISA, Neutralisingmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Recombinant full length protein corresponding to Human Interferon alpha 2b.
    Database link: P01563

  • Positive control
  • General notes

    This product was changed from ascites to tissue culture supernatant on 28/11/2017. Lot numbers higher than GR314306-1 will be from tissue culture supernatant. Please note that the dilutions may need to be adjusted accordingly.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    PBS with 0.1% sodium azide, pH 7.4
  • Concentration information loading...
  • Purity
    Protein A purified
  • Clonality
    Monoclonal
  • Clone number
    9D3
  • Myeloma
    NS0/1
  • Isotype
    IgG1
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab9386 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Sandwich ELISA Use at an assay dependent concentration.

This antibody can be used in a sandwich ELISA for the determination of human IFN-a2b. It recognizes different epitopes than anti-Interferon alpha 2b antibody [4E10] (HRP) (ab5258). We recommend using this antibody as a capture antibody and ab5258 as a detection antibody.

Inhibition Assay 1/400 - 1/1000.
WB 1/1000 - 1/5000.

At 5 µg/ml, this antibody inhibits 90% of IFN-a2b antiviral activity in vitro: The bioassay was performed using L41 cell line and mouse encephalomyocarditis virus (EMCV). Interferon was pre-incubated with the ab9386 for 2 hours at RT and then added to the cell monolayer. 24 hours later the cells were infected with EMCV and cultured for further 24 hours. Antiviral activity of IFN was evaluated using crystal-violet staining. PAbs to respective interferons and an irrelevant MAb of same isotype were used as controls.

Indirect ELISA 1/1000 - 1/10000.
Neutralising Use at an assay dependent concentration.

Target

  • Function
    Produced by macrophages, IFN-alpha have antiviral activities.
  • Sequence similarities
    Belongs to the alpha/beta interferon family.
  • Cellular localization
    Secreted.
  • Information by UniProt
  • Database links
  • Alternative names
    • Alpha 2a interferon antibody
    • IFN alpha 2b antibody
    • IFN-alpha-2 antibody
    • IFNA antibody
    • Ifna2 antibody
    • IFNA2_HUMAN antibody
    • INFA2 antibody
    • Interferon alpha 2 antibody
    • Interferon alpha A antibody
    • Interferon alpha-2 antibody
    • Interferon alpha-A antibody
    • LeIF A antibody
    • LeIFA antibody
    see all

Images

  • The nitrocellulose membrane was blocked for 1 hour at room temperature with 5% milk in TBST buffer. The individual blots were probed  to detect the appropriate model protein for 1 hour at room temperature on a shaker. 

    ab9386 was used at 1 μg/ml diluted in TBST. After briefly washing the blot membranes with TBST, the secondary antibody was added for 1 hour at room temperature with shaking. The secondary goat anti-mouse AP conjugated antibody was diluted 1:10,000 in TBST.

    For full details see figure 11.

  • Standard curve for the determination of human interferon-alpha 2b by two-epitope ELISA: capture antibody 9D3 (ab9386), peroxidase-labeled antibody 4E10 (ab9388).

  • Neutralizing activity of monoclonal antibody 9D3 in vitro. Interferon antiviral bioassay: L41 cells, mouse encephalomyacarditis virus (EMCV). Antibodies at 5 µg/ml. Control 1 - without EMCV (non-infected cells), control 2 - without interferon, control 3 - irrelevant antibody.

References

This product has been referenced in:
  • Landowski CP  et al. Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei. Microb Cell Fact 15:104 (2016). Read more (PubMed: 27287473) »
  • Chen J  et al. Dissolving microneedle-based intradermal delivery of interferon-a-2b. Drug Dev Ind Pharm 42:890-6 (2016). Read more (PubMed: 26467418) »
See all 8 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Answer

Thank you for contacting us. Concentrations of the antibodies ab9386 and ab9388 are determined spectrophotometrically by an adsorbance measurement at 280 nm. The absorbance value (A280) is divided by the extiction coefficient of mouse IgG - 1.35. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for contacting Abcam. For both ab9386 and ab9388, we currently sell them in 100ul aliquots, at a concentration of 2mg/ml. From what I can see, there are no plans to change this concentration for future lots of the antibody and since it is a mouse monoclonal there should not be a large variation in batch to batch concentrations. If any changes do occur then the new concentration will be clearly displayed on the datasheet and/or vial. Please let me know if there is anything else I can help you with.

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Answer

Thank you for your inquiry. Neutralizing activity of ab9386 has been tested in cell line-based bioassay where IFN has been applied in a concentration range between 4 IU/ml and 0.125 IU/ml. If you use lower concentrations of IFN (below 0.125 IU/ml), we suppose that this antibody will efficiently block the activity of IFN as well. If you use higher concentrations of IFN (over 4 IU/ml), we would suggest to determine the blocking activity of ab9386 experimentally by adding this antibody at increasing concentrations (starting from 5 micrograms/ml). I hope this information helps. Please contact me with any other questions.

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Question

BATCH NUMBER 16494 ORDER NUMBER 114870 DESCRIPTION OF THE PROBLEM No signal SAMPLE pure human IFN alpha PRIMARY ANTIBODY 1st set of experiments: PcAb anti IFN alpha from [competitor A] (raised in rabbit) 2nd set of experiments: MAb anti IFN alpha biotin conjugate clone 7N41 [competitor B] Diluent: wash buffer (50mM Tris-HCl, 0.15M NaCl, 0.05% Tween 20, pH 7.5) Dilutions: 1st set of experiments (PcAb)--> 1/500, 1/1000, 1/10000 2nd set of experiments (Mab-biotin) --> 2, 1, 0.5, 0.25 ug/ml Incubation time: 1h at room temperature on the plate shaker Wash step: washed 3 times with wash buffer; soaked each time for 5 min on the plate shaker DETECTION METHOD 1st set of experiments (PcAb): - Plate washed 3 times as previously described except for the last wash done in assay buffer - added pNPP 1mg/ml in assay buffer (50mM Tris-HCl, 1 mM MgCl2, pH 8.0) - measured the results after 15 min,30 min, 45 min, 1h (for some experiments also after 2h) 2nd set of experiments (MAb - biotin): - Plate washed 3 times as previously described - added Streptavidin Alkaline Phosphatase conjugate ([competitor c]) at the suggested dilution (1/500 of a 1mg/ml stock) prepared in wash buffer - Plate incubated at room temperature on the shaker for 30 min - Plate washed 3 times as previously described except for the last wash done in assay buffer - added pNPP 1mg/ml in assay buffer (50mM Tris-HCl, 1 mM MgCl2, pH 8.0) - measured the results after 15 min,30 min, 45 min, 1h POSITIVE AND NEGATIVE CONTROLS USED No positive control (I am using a purified IFN alpha). Negative controls : 1) No coating + IFN 2) Coating no IFN ANTIBODY STORAGE CONDITIONS 1st Ab received (order number: 111851) stored in aliquots at -20C 2nd Ab received (order number: 114870) stored in the fridge at +4C (ELISA performed the next day to delivery) TYPE OF ELISA Sandwich ELISA - ab9386 used as coating antibody COATING WELL Coating buffers used: 1) 10mM Tris-HCl ph 8.5 2) PBS pH 7.0 Coating concentrations: scouted between 8 and 0 ug/ml. Plus used 5 and 10 ug/ml fixed in other experiments. Coated overnight at +4C (plate sealed) BLOCKING CONDITIONS Blocking buffer: pre-prepared BB from Pierce (Tris-HCL buffer) Blocking step: incubation on the plate shaker at room temperature for 1h SECONDARY ANTIBODY Only for 1st set of experiments (PcAb): Anti-rabbit Ab Alkaline Phosphatase conjugated Diluent: wash buffer (50mM Tris-HCl, 0.15M NaCl, 0.05% Tween 20, pH 7.5) Dilution: 1/1000 Incubation time: 1h at room temperature on the plate shaker Wash step: washed 3 times with wash buffer; soaked each time for 5 min on the plate shaker HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 10 HAVE YOU RUN A "NO PRIMARY" CONTROL? No WHAT STEPS HAVE YOU ALTERED? I have used two different coating buffers. I have varyed the coating concentration I have checked different IFN ranges (to check for an eventual hook effect) I have checked both a PcAb and a MAb (biotin) as primary Ab and for each of the systems I have scouted a useful concentration range. I have perfomed a "direct" elisa: Ab coated on the plate and probed with Anti-mouse alkaline phosphatase conjugate to check if the Ab was on the surface and I got a positive result. I have perfomed the ELISA's in duplicate using a different MAB as coating antibody (Clone MMHA-2 , [competitor A]) and the plates with this Ab were giving positive results when I used PBS as coating buffer. This exclude the possiblility of buffer contamination as a possible cause of the absence of signal in the plates coated with 9D3. I have bought a new vial of 9D3 (order 114870) to check if the freezing had denatured the Ab.

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Answer

I'm sorry to hear you are having a problem with ab9386. The use of monoclonal antibodies in sandwich ELISA always requires a careful selection of a suitable pair of antibodies. Antibodies should be complementary, e.g. they should recognize different epitopes and not inhibit each other in antigen binding. The negative result obtained when using mAb 9D3 together with the mAb 7N41(as in the 2nd set of experiments) might be explained in this way. It is not known if these mAbs are complementary. For example, 9D3 recognizes different epitopes than monoclonal antibody 4E10 (ab9388). We recommend using this antibody as a capture antibody and ab9388 as a detection antibody. When working with a sandwich ELISA, it is also important to determine a suitable antigen concentration. We do not know which range of IFN-alpha concentrations you are using. To prove if the system is working properly we would suggest to use IFN-alpha at concentrations of 10-2000 ng/ml. You also did not indicate which IFN-alpha has been tested in a sandwich ELISA. Is it human IFN-alpha 2b? The mAb 9D3 recognizes human IFN-alpha 2b and 2a but does not cross react with human IFN-alpha 5. We have successfully used mAb 9D3 (as a coating mAb) together with mAb 4E10, ab9388 (as an enzyme-labeled mAb). The coating conditions were as follows: 9D3 mAb added at a concentration of 10 micrograms/ml in Na-carbonate buffer (pH 9.5), the plate incubated overnight. You have used slightly different conditions for a coating (either Tris/HCl buffer or PBS) - this might reduce coating efficiency. The best way to prove if the mAb 9D3 is working well with the IFN-alpha is to test it by an indirect ELISA: 1) coat the plate with a purified IFN-alpha (5 micrograms/ml, Na-carbonate buffer, pH 9.5, incubate overnight); 2) block the plate; 3) add 9D3 at dilutions from 1:200 to 1:5000, incubate 1 h at RT; 4) wash the plate, incubate with anti-mouse conjugate and develop as usually. Repeated freezing/thawing might reduce the activity of mAbs, however, it seems not probable that the activity is totally lost. We would suggest: first, to test the mAb 9D3 by an indirect ELISA. If the result is positive, e.g, the mAb is working at dilutions 1:2000 - 1:5000, it can be further tested in a sandwich ELISA. We would suggest: 1) to use coating buffer as indicated above; 2) to use IFN-alpha concentrations in a range of 10 ng/ml to 2 micrograms/ml. Please let me know if this helps and do not hesitate to contact us for further advice.

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Answer

To use monoclonal antibodies 9D3 and 4E10 in sandwich ELISA for the detection of huIFN-alpha-2b, 4E10 antibody should be labeled either with horse-radish peroxidase or biotin. Monoclonal antibody 9D3 should be used as a capture antibody (for plate coating). Before labeling procedure, antibody 4E10 should be dialysed to remove sodium azide (see Abcam protocol Azide Removal by Dialysis). Biotinylation protocols are available on-line (see, for example: www.protocol-online.org or www.drmr.com). Biotinylated 4E10 can be detected using Streptavidin-Peroxidase (widely used reagent, see, for example www.chemicom.com or www.sigmaaldrich.com). Labeling of monoclonal antibody with peroxidase is more complicated procedure than biotinylation (see, for example, HRP Conjugation Kit description at www.4adi.com). After preparation of 4E10 conjugate, it can be used together with 9D3 in sandwich ELISA for the detection of huIFN-alpha-2b. Short protocol is given below: 1. Plate coating: 9D3, 5 mcg/ml in Na-carbonate buffer (pH 9.5), 100 mcl/well, incubation overnight at +4 C; 2. Plate blocking: 2% BSA in PBS, 100 mcl/well, 1 h at RT; 3. Washing: 3 times with PBST (PBS with 0.1% Tween-20); 4. Incubation with IFN standard and test samples: 1 h at RT, 100 mcl/well, dilution buffer – PBST; 5. Washing: 5-6 times with PBST; 6. Incubation with 4E10 conjugate: 1 h at RT, dilution buffer – PBST. If 4E10-biotin is used, further incubation step with Streptavidin-Peroxidase is required; 7. Washing: 8-10 times with PBST; 8. Development of enzymatic reaction using TMB-substrate.

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Answer

Usually, each monoclonal antibody recognises only a single epitope on the antigen because each hybridoma clone is derived from a single B-cell. This antibody reacts well with both native and SDS-denaturated IFN-alpha 2b. Therefore, we suppose that the epitope recognized by this product is linear (it is not disturbed by denaturation).

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