Anti-Pseudomonas antibody (Biotin) (ab68539)

Thank you for your enquiry which has been forwarded to me as my colleague Karin is currently away from the office.

I am pleased to provide below the testing protocol for ab111016 Gram Negative Endotoxin antibody [GNE11-270.3.1] in ELISA.

When I made further enquiries regarding ab68539, I am sorry I was informed that in fact this antibody has not been tested in ELISA. I sincerely apologize for the confusion and error in this case. The datasheet is being amended. If you have already purchased this antibody, we will of course still provide our guarantee for this application for you.

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

Anti-Gram Negative Endotoxin MAb ELISA Protocol



Abbreviations: BSA bovine serum albumin
PBS phosphate-buffered saline
HRP horseradish peroxidase

1. Bacterial cells (5 x 107/ml) are bound to the wells of Immulon II 96-well plates in 50 ul of 100 mM carbonate buffer, pH 9.6, per well. Coated plates are sealed with plastic wrap and incubated overnight at room temperature.

2. The next day, wells are blocked for 30 minutes with 1% BSA-PBS-0.05% Tween 20. This solution is removed by inverting the plates, then anti-endotoxin antibody is added (50 ul/well) for 30 minutes at room temperature with gentle agitation (such as on a rocker platform). Note: customer should test a range of dilutions/concentrations of endotoxin antibody in order to determine the optimal antibody concentration for their preparations of bacteria.

3. Wash plates 3x with PBS-0.05% Tween 20 by filling all wells then inverting plates. Remove as much wash buffer as possible between each wash.

4. Secondary antibody, anti-mouse IgG/IgA/IgM-HRP is diluted in 1% BSA-PBS-0.05% Tween 20. Customer should determine the optimal dilution for their secondary antibody. The reagent we currently use is diluted 1:800-1:1,000 for use. Diluted secondary antibody is added to each well (50ul/well) for 30 minutes at room temperature with gentle agitation.

5. Wash plates 3x with PBS-0.05% Tween 20. Remove as much wash buffer as possible between each wash.

6. To each well, add 200ul of substrate-chromagen solution of choice followed by incubation suitable for the selected substrate-chromagen. Add stop solution if required and obtain optical density (O.D.) readings.

An ELISA using this antibody can also be set up for purified LPS coated on the microwells. Purified LPS’s are available from commercial sources. They are provided lyophilized, and we reconstitute them to 1-2 mg/ml in PBS, pH 7.4. Aliquots are stored at -20oC. Dilute LPS to 20 ug/ml in a DOCA-containing buffer for coating wells:

DOCA Buffer
0.1 M phosphate/NaCl
2.28 g monobasic sodium phosphate
11.5 g dibasic sodium phosphate
163.0 mg NaCl
0.882 g deoxycholic acid
Bring to 1 liter with distilled/deionized H2O.

The remainder of the protocol is as described above.

Thank you for contacting us. I apologize for the delays.

The antibody was raised against Pseudomonas (whole cells) and is a polyclonal specific for Pseudomonas. Because whole cells were used as the immunogen to generate this polyclonal antibody, because many bacteria have common cell surface antigens, and because the antibody is specific for a number of epitopes (being polyclonal), it has been shown (and is not surprising) that this antibody can cross-react with other bacteria.

However, this does not mean the specificity of the antibody has changed or that it can be considered an antibody specific for Gram-negative bacteria. The cross-reactivity is likely due to the fact that the antibody has not been pre-absorbed.In fact, it has been tested against other Gram negative bacteria by IFand shown to react, so it is not surprising to me thatyou observes binding to other bacterial species

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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Thank you for contacting us.

As discussed please send me the details e.g.how the antibody was used.

I will be looking forward to hearing from you soon.