First check at your side that polyclonal antibody in rabbit is IgG or IgM. If it is IgM than the problem will come. IgM is pentamer so it will increase the size of nano-particle and by virtue of increase in size it will not flow. Secondly you may carryout the scanning of absorption spectrum of conjugated and non-conjugated (poly clonal and monoclonal antibody coated) gold nanoparticles (NPs) spectro-photometrically in the wavelength range of 450-600 nm which will give indication of nano-particle size. 

Hi Tulsidas,

Thank you very much for your response. The polyclonol antibody is IgG. Will check again. Will do the absorption spectroscopy as you suggested to know the increase in size. You feel that size of the conjugate can be the factor to consider?

Thank you for your time and thoughts

I would definitely check the size of the particles generated.  The other thing to consider is your antigen.  How many potential epitopes are being presented to the antibody.  The monoclonal is probably more likely to produce a smaller complex due to self competition which depends on the monoclonal concentration.  Polyclonal antibodies will exist in a range of concentrations for any given clone within the polyclonal.  Clones to different epitopes will almost certainly not be evenly represented.  This may cause the problems you are seeing.  If you are at antigen:antibody equivalence for any given clone you will maximize complex size.  My guess is with the monoclonal you are in antibody excess.  Try diluting the polyclonal.  You may be able to find a dilution that give you appropriate sensitivity without to big a hit to functionality.

Do you see any changes in the colour of the gold with the polyclonal? If you do you could be getting aggregation during conjugation. Usually (but not always) a competitive assay is done because the analyte is small and has only one epitope; antigen induced aggregation is thus unlikely.  An absorbance scan may be required to see aggregation of the conjugate, and it is correlated with a significant increase in absorbance in the 650 nm region. If the aggregation is very significant you don't really need an absorbance scan, the particles will look blue/purple. 

Monoclonal and polyclonal antibodies behave differently with goldnanoparticles at different pH conditions. Perform flocculation assay for the optimization of polyclonal antibodies concentration with goldnanoparticles .  O.D. peak at 530 you should get to work. 

Might be in case of polyclonal antibodies with antigen having more epitopes it is causing stearic hinderece and the system is not working. It is best to conjugate monoclonal antibodies with gold nano particles. If u increase the pore size of NC membrane to overcome polyclonal antibodies behave u will get reduced time for antigen antibody reaction time which subsequently hamper ur sensitivity.

It may well be that the conjugate with the polycloncal is simply not stable, i.e. insufficiently protected against salt challenge.  All you need to do is try various concentrations of antibody at various pH's in order to get a stable conjugate.

I too am suspecting suboptimal conditions for your PAb conjugation.  It may require a different pH than the monoclonal conjugation and different coating concentration.  What happens is an unstable conjugation aggregates on the pad, and this aggregation results in the conjugate not flowing out of the pad.  Based on what you're describing, this is exactly what is happening.  IgM complexed to the gold is not an issue, it should not make the particle size so large it will not flow.  There are many commercial IgM lateral flow assays out there, including one of my own.

Hi James Drummand,

Thank you very much fro your response, It is helpful to know the information you said.

I will certainly dilute the concentration and try out again with polyclonal. Thank you very much

Hi Nick Gee,

Thank you very much for your response. I did not see any visible colour change to blue/purple. But I didn't check the UV-Vis absorbance. That would give me an idea of the size of the conjugate as you suggested. I will measure the absorbance and check the size. Thank you so much

Hi Jyothi Singh,

Thank you very much for your response. Your suggestions are helpful. I have not done the flocculation assay to optimise the concentration. I have optimised for monoclonal used the same concentration as that of monoclonal for polyclonal too. I will perform the optimisation assay for polyclonal and try out again as you suggested. I will not reduce the pore size as I don't want to compromise on the sensitivity. I am trying polyclonal to make the assay less expensive. Thank you again for your suggestions

Hi Josh Lizer,

Thank you so  much for your response. As you suggested, size may be the factor. I did not optimize the concentration of Poly Ab. I will do the optimisation step and repeat. The antibody I am using is an IgG and not an IgM. 

Thank you again

Have you tested whether both the antibodies work identical using any other assay system?