Anti-Bradykinin抗体(ab47686)

Thank you for your reply.


I apologize for the error on our datasheet for ab47686. As requested, I have placed an order free of charge for the Bradykinin antibody ab14391.


Below please find the ELISA protocol:


Indirect ELISA Protocol


A. Materials required:

1. primary antibody
2. Conjugated secondary antibody (HRP or biotin, etc.)
3. Colorimetric substrate (such as TMB, OPD, enzyme-linked avidin)
4. Coating buffer: 50 mM sodium carbonate, pH 9.5
5. 1X PBS (Phosphate buffered saline): 8.0g sodium chloride, 1.3g dibasic sodium phosphate, 0.2g monobasic sodium phosphate in 1.0 liter distilled water, pH 7.4.
6. 1X PBS-T (Phosphate buffered saline-Tween 20 solution (PBS-T): PBS containing 0.05% Tween-20.
7. Non-fat dry milk.
8. Blocking buffer: PBS-T, 5% non-fat dry milk.
9. ELISA 96 well microtiter plate or 8 well strips.
10. 2N HCl


B. Antigen Application

1. Add 100ul antigen solution and serial dilutions of standard (if available) diluted in coating buffer to appropriate numbers of microtiter wells.

2. Incubate at 4°C overnight (or 2 hours at RT) in a humid environment, i.e, covered by a glass plate or in a sealed box with a dampened paper towel inside.

3. Empty microtiter wells and invert plate to tap out excess liquid onto a clean tissue.


C. Blocking Step

4. Add 200-300ul of blocking solution to each well.

5. Incubate 1-2 hours at RT, empty plate and tap out excess fluid onto a clean tissue.

6. Wash wells three times with PBS-T.


D. Primary antibody Incubation

7. Add 100ul of primary antibody solution diluted in blocking buffer to each well (1:300).

8. Incubate 1-2 hours at RT (or 4 hours at 4°C) with gentle agitation (on a rocker plate, for example).

9. Invert plate and tap out excess liquid onto a clean tissue.

10. Wash wells three times with PBS-T.


E. Secondary antibody incubation

11. Add 100ul of secondary antibody diluted in blocking buffer to each well (according to manufacturers instructions).

12. Incubate 1-2 hours at RT with gentle agitation.

13. Invert plate and tap out excess liquid onto a clean tissue.


F. Wash Microtiter Wells

14. Fill each well with Wash solution (PBS-T), agitate 5 min at RT.

15. Invert plate to empty and tap out residual fluid onto a clean tissue.

16. Repeat wash step 3 times.


G. Color development

17. Add TMB Substrate to each well. Seal with tape and incubate the plate for 15-30 minutes at RT.

18. Add STOP Solution (2N hydrochloric acid or 2N sulfuric acid) to each well. Shake gently for a few seconds. NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.

19. Read absorbance at 450 nm with microplate reader within 30 minutes after adding STOP Solution.



I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions.

Thank you for your patience as I have looked into your inquiry further.


It seems that this product has not be validated for ELISA, but rather competition assays so unfortunately we do not have specific protocol conditions to recommend. I would be happy to replace this product with a Bradykinin antibody that has been validated for ELISA, such as ab14391.


I apologize for the inconvenience caused. Please do not hesitate to contact me if you have any additional questions.

Thank you for your inquiry.

For the three of our bradykinin antibodies, ab47864, ab14391and ab47686, the immunogen used was from a.a. 381- 389 of the human sequence (RPPGFSPFR).

You can see any testing information on the online datasheets:

https://www.abcam.com/Bradykinin-antibody-Biotin-ab47864.html

https://www.abcam.com/Bradykinin-antibody-ab14391.html

https://www.abcam.com/Bradykinin-antibody-ab47686.html

I hope this information helps. Please contact us with any other questions.