Can this be due to the way the primary AB is being immobilized that the secondary is not finding free binding epitopes after the proteins in the lysate have bound the primary? If so the possible solution can be either immobilizing thre secondary AB and incubating with a mixture of primary Ab and lysate (basically the antigen-antibody mixture) or using a capture Ab which can act as both primary and secondary.

First: Are the antibodies poly or monoclonal? What are their sources? Do you know exactly the epitope for each antibody? What are the dilutions/concentrations you are using in each case?

I would recommend to do Dot/Western blot for each antibody to confirm recognition.

Logic would suggest that if you are using the same antibody immobilised, and for detection, then you are binding to the epitope with the immobilised antibody, and the detection antibody has no epitope to bind to. Could you use a different detection antibody to the same protein, but raised against a different epitope (preferably not one masked by the first antibody).

Use a monoclonal antibody for capture and a polyclonal antibody for detection. In addition, make a titration of both the capture Ab and the detection Ab, maybe the concentration is too low?

Dear James,

there are quite a few reasons for your observations.

how did you select the antibody pairs?

Have you looked for matched pairs?

The assay buffer seems suitable. My guess it is a steric hindrance. It is probable that the 2nd ab doesn´t bind due to epitope interference. Another reason could be that the abs are not suitable for ELISA. Look for application IP tested. If the abs work in an IP they should work in a sandwich set-up. In WB the proteins are denatured for a sandwich assay it is a prerequisite taht they recognize their anrtigen in a native form.

How do you detect the binding of the capture ab? By SPR?

best Olli

I agree with the assumption that the two antibodies might not match. It is not trivial to find a good matching pair, e.g. http://www.ncbi.nlm.nih.gov/pubmed/16284655

I agree with Ingvild suggestion. Use a combination of mono and poly antibodies. That may work. Other thing , the concentration of your secondary antibody (2 ug/ml) is quite high, that might be creating a crowding effect. Have you tried different dilutions of your secondary antibody?

Are you sure that your second antibody is still working when labelled?

This question is very similar to that of Omar J BenMarzouk-Hidalgo posted Sept 3, 13. You might check the 39 answers given there.

Best wishes!

Hello. There could be several reasons why your sandwich assay is not working. As mentioned above, are your antibodies monoclonal or polyclonal? If monoclonal, could it be that they are binding to overlapping or competing epitopes? Also, sometimes steric hindrance can be a problem with binding of one antibody, stopping the other from binding as expected. Have you tried turning the assay upside down as in use the secondary as a capture antibody and detect with the one you were using initially (I am of course assuming that neither antibodies are labelled) and then you could detect with labelled anti-species antibody? This is how I solved an ELISA issue, I had once. The assay will work one way but not the other! I am also assuming that you have tested the antibodies individually and know that they are binding to target of interest (maybe using recombinant protein or partially purified prep?). Since, you are looking at lysate which contain a mixture of potential targets, are there any possibilities that cross reactivity (maybe with detection antibody) being an issue? Another approach, is to allow binding of secondary to target to take place in solution externally (tube/well) and then add the antibody-antigen complex to your capture antibody. This will hopefully allows the complex to be pulled out of solution so that you can detect it but might not be practical in a multiplex assay. It will however answer the question as to whether the assay might work.

I’m afraid that I cannot answer the question directly, but hope that some of my thoughts might be useful. All the best.

In this case, check secondary ab ' species specifity,and try to use the secondary ab (conjugated ab) the same species specifity as the primary ab ( capture ab) ,for example, goat anti mouse for the primary and secondary abs.

If you are developing multiplexed assay, what are you capturing and how many proteins are you trying to detect at once? If you are capturing a mix of proteins and detecting a few at a time, you might be below sensitivity level. Although your detection antibody is binding you are not able to detect your specific protein because a few proteins are captured. Try to figure out the problem with the specific capturing of one protein and detection and then proceed to the mixed capture and further detection. Capture antibody has to have very good off-rate to be able to keep your protein of interest. Also, as it was mentioned, make sure that capture antibody does not have an overlapping epitope with the detection one.

Your original question mentions that you are developing a mutliplex assay. Are none of your target proteins being detected? If so, you might want to look at your lysate preparation. If it is only one or two than it is most likely a poor choice of antibody pairs.

Another thing to consider whey probing lysates is that many cellular proteins are already part of complexes. These complexes may be interfering with one or more of your antibody pairs. You need to understand the biology of what you are after to determine if this is a problem.

Instead of "bad" I would rather refer to the question of suitability of ABs and methods for the desired results. To start with a few questions:

1. are you using polyclonals or monoclonals

2. are capture and detection antibodies the same, from different animals and against the same target protein

3. is the detection AB labelled or how is the detection of AB binding planned Surface resonance or what else.

What do you know about the reactivities of your antibodies for example:

a influence of binding to solid surfaces to reaction with antigen

b influence of label to binding specificity

c interference of the two antibodies also in relation to first and second antibody in your test design

These question should be made always with the same protein prep when possible and a positive as well as negative control. Otherwise the determination of reasons for negative results and the achievement for robus results is not possible.

Now for starters of methods choices:

1. I understand you worked or must work with a DAS- or TAS-design and the label of ABs is an enzyme.

Is a plate trapped antigen format possible for you? This would allow the use of unlabelled ABs for detection in combination with a commercial final antibody for the determination of bound antibodies. It would ensure the full serological reactivity of the specific AB. For a multiplex assay you need to confirm for each different specific AB its suitability and the optimal concentrations. In this case it seems appropriate to keep the types of ABs, i.e. poly- versus monoclonal at a minimum.

In Summary, make sure you know as much as possible about the ABs then choose the most suitable detection method and format of assay. just for consideration. Instead of an ELISA- like design on plates you may look at nitrocellulose as solid surface or even plastic beeds, this might have an advantage in reducing material needed and handling time and sensitivity.

when you have more detailed information dont hesitate to ask more specific questions. Nothing to worry I am retired already.

Determining what all needs to be corrected is most important. Suggestions from the comments above would certainly help in solving the problem but too much of experimentation randomly done is often confusing. A thorough study of every step is essential before a practical experiment.The planning of the very first experiment should be such that you must get a positive outcome. The assay can be improved thereafter.

Hi! May be it will be interesting for you. https://www.researchgate.net/publication/228106684_Analysis_of_protein-protein_interactions_in_a_complex_environment_capture_of_an_analyte-receptor_complex_with_standard_additions_of_the_receptor_(CARSAR)_approach?ev=prf_pub

Article Analysis of protein-protein interactions in a complex enviro...

I agree with Dr. Deuskar. Add a positive control to your experiment that "must" work. Then you can trace down the problem 1 step at a time. You may be dealing with an issue with how the first antibody is coating the surface - if it lays down on the surface you may never see any reaction no matter what else you do...

Good luck!

Ryan.

This first binding of analyte molecule to primer antibody; are you sure that is a binding or adsorption by lysate compounds?

Dear James,

if you are still struggling with the setup of you experiment i would like to suggest you to check the Attana instrument http://www.attana.com/

Compered to an ELISA assay you would be able to follow in real-time the binding events and to minimize non-specific interactions thanks to the specifically designed chip surface. Furthermore cell-lysate or serum can be directly injected in the system without worrying about clogging problems.