Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (AP) (ab6722)

I repeated the experiment and diluted plasma samples out to 1:4800 fold but this dilution dependent signal increase phenomenon persisted. In other words, the signal from the 1:4800 fold dilution was higher than the 1:2 dilution! I really doubt if this is a hook effect as I would expect the signal to decrease again after adequate dilution and not continue to rise.

Then I thought whether it could be non-specific binding of detection ab (I used ab6722 - a Goat anti-rabbit ab because my primary is Rabbit anti-human). So I included controls in which I added only the secondary ab to plasma-coated wells, without any primary. I saw moderate signal from these wells (ie higher than BSA negative controls but weaker than corresponding plasma-coated wells with both primary and secondary added). Does this mean the secondary ab is binding non-specifically to something in the human plasma? Are you aware of cross-reacitivity of this Goat anti-rabbit ab (ab6722) with any human Ig??

ANSWER:

I am not aware of any cross reactivity of this antibody with human Ig. However we do offer a number of secondary antibodies which are designed to reduce background levels by either being pre-absorbed against other proteins and Igs or by digestion into F(ab) and Fc fragments. I've included links to a few of those below.

That said I do not feel that the background you are experiencing should be the cause for the type of response you have been seeing. This is usually something that can be easily subtracted from your results if you run 2 or 3 wells with this control as a baseline.

Another possibility is that it could be that the dilution buffer you are using is somehow causing this reaction. What buffer are you using? Have you tried using filtering this or using another buffer?

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

These are links to the antibodies that have been pre-absorbed:

http://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-Alkaline-Phosphatase-pre-adsorbed-ab97072.html

http://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-Alkaline-Phosphatase-pre-adsorbed-ab97072.html

http://www.abcam.com/Goat-F-ab-2-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-AP-pre-adsorbed-ab98469.html

Many thanks for your reply. I really appreciate your suggestions. My detection is working well now. However I am facing some strange results which I would love your thoughts on please:   I am struggling to interpret an indirect ELISA I am making. It involved coating wells with human plasma overnight, blocked with milk the next day, then primary ab (Abcam), then detection ab (Abcam) and finally the ALP substrate.

I ran parallel wells in which I coated with   1)      known concentration of antigen which is a recombinant protein to generate my standard curve and 2)      BSA (negative control) I got nice reproducible standard curves (R2=0.99) and uniformly negative signals from BSA. So all good.

However I am seeing a dose-dependent increase in signal in plasma samples. As plasma dilution increased, signal increased - in other words, the more dilute the plasma (so in theory lesser amount of antigen) - the stronger the signal! I could not understand why. I repeated the experiments and I am seeing it every time (4x now).

At first I thought it could be due to primary ab binding to "unoccupied sites in the wells" directly, which then generated a signal when secondary added, which would explain why the signal is stronger in diluted sample. But if that's the case I should see that in the BSA wells, but I am not. I did block the wells with milk after coating to ensure all unbound sites are occupied. Then I thought could it be something in the plasma which is inhibiting the substrate reaction (ALP). So the more dilute the plasma, the less the inhibition, and the stronger the signal. But plasma was only used to coat the plate overnight. The plates were then washed after every step with TBS-tween (2-4 times) after every step. So I would imagine not much of any “inhibitors” are around by the time substrate is added.

Then I thought whether it could be a “hook” effect. In other words, the plasma is too concentrated and antigen are crowding up thus reducing the amount of binding. But I thought usually plasma then need to be diluted up to 100 folds to relieve the hook. But I am seeing a “nice” dose dependent increase in signal as plasma was diluted from 1:2, 1:4, 1:8 to 1:16 only.

ANSWER:

Thank you for contacting me again. It is always a pleasure to hear from you.

After reviewing your email, I am lead to believe that this really is a hook effect as you had mentioned. I am not an expert on this hook effect in plasma specifically, and so cannot comment on the 100 fold dilutions usually required, but your description does sound like a hook effect. These should eventually tail off with further dilutions.

The hook effect may also be created by overcoating with primary antibody; you may wish to further dilute your primary to tackle this as well.

I hope these suggestion help. Let me know if you have any questions.

Thanks for your advice and suggestions last time. I got another ab7468 and it is working well, both for WB and my reattempt ELISA.   However I have a question which I hope you may have an answer to:   After adding the pNPP substrate, I waited for 30 min, as recommended, but the signal was very weak. OD was around 0.1 and negative control 0.06. However as I waited for longer, up to 1.5 hour, the signal intensified in the samples and went up to about 0.4. Negative control still remained low at 0.06, which was good. The resultant standard curve was great (R2=0.99). However I could not understand why it took almost 60-90 min before the OD got up to a “good signal”. I don’t think I am simply increasing background as the standard curve was good and the longer duration did not intensify signal from any of the negative controls.   Is this longer than usual duration due to 1)      Inadequate primary ab? 2)      Inadequate secondary ab? 3)      Inadequate coating antigen on plate??  

ANSWER:

Thank you for contacting us, I am glad to hear that the new product is working well for you.

Usually when color is developing slowly it is one of three issues. 1) the plates and reagents have not come to room temperature. 2) the conjugates are weak, try to prepare these right before use and at correct concentration 3) the presence of peroxidases, azide or other contaminants are interfering with the reaction, these may be removed by dyalisis, gel filtration or with purification columns.

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

Do I need to add a stop solution (ie NaOH) at the end of the sandwich ELISA after the colour develops with ALP chromogen or that is not necessary?

ANSWER:

Thank you for contacting us.

Just as with a common indirect ELISA it is important to stop the reaction using the appropriate stop solution.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

I recently purchased your ab (ie Rabbit anti-human FNDC5 IgG), to be used in a ELISA. I am wondering what detection system would you recommend?   Ie According to the Abcam ELISA protocol, one option is HRP.   So Do I add a secondary ab conjugated (ie anti-rabbit IgG Fc, HRP conjugated), followed by washing and then dispensing 100 ul of substrate solution (ie TMB?)   Or would you recommend other secondary/substrate solution system?  

ANSWER:

Thank you for contacting Abcam.

Enzymatic detection with an antibody that has been conjugated to HRP or to AP is a widely used and robust method. As this target is located on the peroxisome membrane I would recommend using anAlkaline Phosphataseconjugated secondary for this target prehaps ab6722,Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (AP). Followed by an appropriate substrate detection of your choice. I recommend theAlkaline Phosphatase chromogen (BCIP/NBT). We do offer this as a ready to use product, ab7468.

I hope you have found this information helpful. Please let me know if you have any questions. I have places links to each of the products I have mentioned here below.

Ab6722:http://www.abcam.com/Goat-polyclonal-Secondary-Antibody-to-Rabbit-IgG-H-L-AP-ab6722.html

Ab7468:http://www.abcam.com/Alkaline-Phosp