Hello, me again~

you coated secondary antibody and use a gold nano-particles labeled primary antibody for detection.

the secondary antibody would randomly immobilized on NC membrane (usually physical absorption) , so they could be any orientation. The secondary antibody use its variable region to recognize the Fc region of primary antibody which is conjugate with nano-particles here.  The conjugated nano particles may affect the primary-secondary antibody recognition more or less, that result in low binding or even not binding at all.

if you can tell me more specific details about your experiment and your purpose, maybe i can give you more advice.

Is your secondary antibody and anti-Fc, anti-Fab, anti-whole IgG? anti-gamma? This can also influence the ability to recognize gold-conjugated antibodies...

Hello Yudung Lu,

Thank you very much for your answers and your willingness to help me. Thank you.

As I was telling, I have to detect a small molecule called guanosine using a paper sensor. So I was using AuNP conjugated antibody for detecting that molecule. I have spotted BSA-guanosine (to make the moeity big, as guanosine itself is too small) on a Nitrocelluose membrane (NC). Then blocked with 2% milk once, another time used 2% BSA. Then dipped the spotted strip in the solution of AuNP-antibody. The paper didn't show any colour, either the spot or the rest of the paper. When I did a control without blocking, the rest of the paper showed colour but not the spotted regions. I could not figure out what an be the exact reason.

To test whether antibody bound to AuNPs is having functional integrity, I have done this experiment of coating secondary antibody and then coming up with AuNP-primary antibody conjugate, to check whether the primary antibody coated with AuNP is able to bind the secondary antibody. I have used 0.75 % milk as blcoking agent.

I found that only the spotted region showed no colour, while the rest of the strip is coloured. 

What can you figure out ? What would have went wrong ?

Thank you very much for your time.

Hi Gertrudis,

Thank you very much for your response. Here are the details of the antibody.

Antibody Format: Whole IgG

Specificity : IgG (H+L)

Minimal Cross Reactivity: Bovine, Horse, Mouse Serum Proteins

Conjugate: Unconjugated

Product Category: Whole IgG Affinity-Purified Antibodies

Clonality: Polyclonal.

Will it work based on  the information given above ?

Thank you for your time

Hi Abilash

It's my pleasure.

I think i can undersdand your purpose. you want to use a immobilized secondary antibody bind to the AuNP-antibody to confirm that the AuNP-antibody is functional.

Here i need to clarify the antibdoy you used.

according to your description, your primary antibody should be a XX-anti-guanosine antibody(XX means the antibody origin species,eg.mouse/human) and your second antibody should be a YY-anti-XX antibody (YY is the species of secondary antibody , XX is the species of primary antibody, eg. Mouse-anti-human antibody). if it is all right. The AuNP-conjugate could affect the two antibody binding as i said, so your confirm experiment may be fail for this reason.

I think maybe you can try to add different concentration of BSA-guanosine into AuNP-antibody solution to see if it can cause immunoprecipitation.

Hi Yudung,

Thank you for your response.

Primary antibody is rabbit anti guanosine, the secondary antibody is mouse anti-rabbit.

Will it work ?

How do i detect using immunoprecipitation ?

Thank you for your responses

I f your secondary antibody is a polyclonal antibody recognizing both rabbit H and L chains, as I understood,  it should in principle recognize your rabbit antibody conjugated to gold. This is a good control. Conjugation can destroy some epitopes, but should not destroy every epitope recognized by a polyclonal antibody on H and L chains of every conjugated molecule.

Are you sure that the secondary antibody was obtained in mouse? This has no influence on the results, but it sounded strange that its from mouse and is a polyclonal one. Usually the polyclonalas are obtained from big animals producing larger amounts of serum, If it is monoclonal, the answer would be different, because its specific epitope could have been affected by conjugation. If its actually polyclonal, your control should work.

Perhaps the antibody was not conjugated to Au, the conjugation process could have failed.

Sometimes when gold staining is not sensitive enough to produce a visible signal, silver enhancement can produce a stronger signal. This is an excellent amplification system for gold.   

It seems like your antibody is no problem.

sorry for that, i actually mean "Immunoturbidimetric"  instead of " immunoprecipitation".

You could ad different titer of AuNP-IgG into BSA-guanosine, see if it could cause a preciitation to insure the binding. But i am not very sure weather it could work for labeled IgG. you can have a try. 

Here is a paper for reference:

Hi Gertudis,

I just looked back. The secondary antibody is from goat. It is Goat-anti-rabbit antibody. Sorry for the wrong information. As you suggested, I will have to check for the confirmation of conjugation of antibody to AuNP. I will also look into the process of silver enhancement. Thanks for the information. It has given some hope.

Hi Yudong,

Thank you for your response. I  started looking into papers on Immunoturbidimetric assays using nanoparticles. Will see if it can be done.  Thank you.

If you get any progress or figure out what's wrong with your experiments, hope you can notice me. I am also interested in this question. Thank you~

I will do that surely Yudong.

Thank you so much. Will be further needing your suggestions and advice may be.