Anti-Interferon alpha 2b antibody [9D3] (ab9386)

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.

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.

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.

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.

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.

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).