It depends on the virus you use. You had better specify the virus you use, as well as the details of your experiment.

I would suggest trying a depletion experiment. Incubate the virus particles with different concentraions of your (monoclonal?) antibody. Subsequently perform an ultracentrifugation to spin down the viruses. Subsequently determine the mab concentrations in the supernatant (SN) with a suited highly precise and accurate immunoassay or other suited method. The concnentrations of the binding partners need to be carefully adjusted in order to saturate the viruses but neverthelss observe a significant depletion of the mabs in the SN. This will definietly need a lot of optimization and appropriate controls - and your mab should have a fairly high affinity. From the reduction of the mab concentration in the supernatent you may determine the bound mab portion. In order to get a 1:1 binding you may consider using Fab instead of complete IgG.

Thanks! I am working with papillomavirus focusing on structure change before and after binding with different monoclonal antibodies and corresponding Fabs. We are able to produce highly purified virus particles but have not an accurate way to determine the quantity of the virus particles. The same with the mAbs and Fabs that bind with the virus particles. We can tell the concentration by either using OD280 or protein assay. But it is not enough. I hope there are some advanced technologies that can be used for this purpose.

Maybe you could analyse the mAbs that remain in the supernatant and just make a mass balance.

Try a RAPID ELISA. It's the fastest and easiest. Follow this link which I used for Rotavirus..it should help you.

http://icmr.nic.in/ijmr/2004/0202.pdf

Search for similar test development methods through Google.

Thanks for your answers! I guess my question was not articulated clearly enough. We really want to have a way to find out the ratio of virus particles/monoclonal antibody molecules at binding. I came across a virus particle counting machine, did anybody use it before?

look at this

http://www.bitplane.com/go/knowledge-base/case-studies/4d-quantitative-analyses-of-virus-infection?utm_medium=email&utm_campaign=Newsletter%20Q3%202012%20-%20Region%20ROW&utm_content=Newsletter%20Q3%202012%20-%20Region%20ROW+CID_21321fb779d0a7953749bd2183661c30&utm_source=CampaignMonitor&utm_term=4D%20Quantitative%20Analyses%20of%20Virus%20Infection

One approach if you have the technology available or could find a suitable CRO would be SPR. Immobilizing your antibody on the surface, capturing the virus and then coming in with more antibody. You would want a low density of antibody on the surface to minimize crosslinking via one virus particle. The response on the surface is directly proportional to mass. So the response you get when attempting to saturate the captured antibody with additional antibody can be directly related to the amount of antibody bound to virus. For example, assuming the ratio of mass between mAb:virus is 1:10. Let's say you manage to capture 100 response units of virus on the surface. Assuming only one additional antibody could bind per captured virus the maximum response you would expect under those circumstances is 10. If you flow the antibody over this captured virus at saturating concentrations and the response you get is actually 60 that would indicate 7 (6+1 for the capture antibody) bound to your virus. You can vary the surface density of capture antibody to ensure you are not getting cross linking and as long as the capture surface is stable and there is a strong interaction between your antibody and virus this should work and require minimal sample.

If this approach is not plausible for you then Bodo's suggestion of a depletion experiment could work.

Screen a pair of monoclonal antibody which can bind 2 different site.. So you can quantify by sandwich elisa.

Have you money to buy a peice of kit??? These are very sensitive and I think will help you determine the parameters you require;

http://www.izon.com/products/qViro-X/

Ted Pierson at NIH in collaboration with Mike Diamond at WUSTL published a study in Cell Host & Microbe a few years ago that addresses this issue directly, finding that as few as 30 antibody molecules could neutralize a WNV virion: http://www.cell.com/cell-host-microbe/abstract/S1931-3128(07)00040-6 also free here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2656919/

They achieved this using functional avidity estimates as well as clever use of a mutant envelope protein in various ratios with wild type. Perhaps, if analogous tools are available for HPV you can do something similar. Ted Pierson is also a really nice guy, and may be of some assistance.

For quantification of virus particle, there are two commonly used methods: plaque forming unit and 50% Tissue and Cell Infection Dose. Also there are many resources to find these protocols applicable to your virus. For your study purpose, I'd suggest plaque forming unit (PFU)assay as 1 PFU usually means 1 virus particle. Hope this helps.

It seems to me that you need to have an accurate determination of your viral particle concentration first of all. Infectivity assay is no good because of variable infectivity to particle ratio in viral preparations. If you are sure all your particles contains DNA (i.e., no 'empty' viral capsids, as in gradient centrifugation during viral purification), your can use the digital PCR technology, based on Poisson distribution, to have an accurate count. If that is not available, you may have to use electron microscopy: mix your virus with a known concentration of 100 nm latex particles, determine the ratio under EM with negative staining, and calculate the virus concentration that way. With a known viral particle input, I suspect the best way to determine the number of ab molecules binding is by calorimetry, if that kind of instrumentation is available to you.

Thank all of you for the helpful messages. We contacted Izon and found out the equipment was too expensive for us. I am wondering if any place owns that equipment and offers service to customers. At the same time, we will try EM reference particle counting which is a method we used before. Once we can quantify the virus particles, we can determine how many monoclonal molecules are sufficient to trigger a visible structural change under CryoEM.