What kind of activity are you looking for? inhibition of viral replication? infection? syncitia formation? What kind of virus? What kind of cells does it infect?

i have purified bio-active protein/peptides and want to test antiviral activity of the same against baculoviruses like nuclear polyhedrosis virus (budded virus) and granulovirus. The strategy i need to employ is to test for the precipitation in agglutination (microwell) plates. I need to observe the existence of resulting precipitate at bottom of wells. I will be serially diluting my samples (two-fold) in appropriate buffer and add equal volume of the virus.

I need to know whether i will be able to observe the precipitate (qualitative) and how do i measure the precipitation % (whether by ELISA)?

If I understand you correctly, the only players in your experiment are the peptide and the virus and addition of the peptide into virus-containing vial should result in viral agglutination and precipitation.

Viral agglutination can be monitored by Electron microscope. Possibly, other methods also available (fluorescently labeled virus?), but I am not familiar with them. You should perform a deep search in the literature. ELISA and other downstream precedures (possibly SDS PAGE will work?) are possible only if you succeed to get rid of soluble, non-aggregated particles (centrifugation? ultra-centrifugation?). However, it is not clear whether viral antigens, recognized by ELISA-related Abs will be available following viral agglutination. Dot blot analyzes with polyclonal Ab will be alternative choice to analyze viral amounts in precipitate.

Quantification can be done by determining the maximum dilution that gives agglutination. (Note, addition of very high peptide concentrations may lead to "prozone effect"). To have a 100% precipitation reference you should use some "gold standard" chemical. Possibly, PEG.

These are my very intuitive thoughts, but possibly they will be useful to start with.

Thanks for your comments. I can therefore summarize it as follows:

1. Qualitative assessment of viral precipitation by microscopy. Actually we have EGFP expressed virus, to be used for the experiment.

2. Quantitative measurement by ELISA or dot blot

Maximum dilution giving agglutination would then represent the peptide titre. Is it so. I would like to know,that if i try to make a curve by plotting time of incubation and precipitation %, then shall i select the maximum dilution viral titre as the standard concentration.

1. Possibly, aggregates of the fluorescently labeled virus will be detected by fluorescent microscopy - depends on the size of the aggregate and on viral amounts.

2. I am not sure I understood your second question regarding the maximum dilution viral titer. Addition of different peptide concentrations (prepared by serial dilutions) into the virus-containing vial will change peptide:virus ratio. Changes in peptide:virus ratio will have an effect on agglutination degree. No dilution of virus is required.

Thanks again. I have conducted the assay with my sample. I got the precipitated virus in higher dilutions of my sample and also in my negative control (virus and buffer), but at half dilution of my sample, i cannot see the precipitate as a button in the microwell plates, rather i can visualize the aggregates of the viral particles. Does this mean my sample has virus inactivating properties? Please give your valuable opinion.

It seems so.

I suppose that you made gross visualization of both precipitation and aggregation. I would add microscopic observations in order to understand how does the precipitation in the negative control look like. I assume that it happened because of pH changes (as a result of addition of peptide-related buffer (w/o a peptide)), or because the buffer contained some linking chemical).

In addition, please provide exact definitions for "precipitation" and "aggregation". Some scientists suggest that they are very close. For example, look at the following link:

http://www.life.umd.edu/classroom/bsci423/song/Lab6.html

Furthermore, you should analyze a wide range of virus-peptide ratios in order to define an "equivalent zone". Equivalent zone is "The range of optimal concentrations, at which the greatest amount of precipitation (or aggregation in your case) is generated". Very high concentrations of peptide or, alternatively, very high concentrations of the virus will inhibit formation of the aggregates.

And the last thing. Possibly, the paper below will help you since it uses similar methods.

http://ajplung.physiology.org/content/273/6/L1156.full

Good luck.

Hope this information will help you and I hope that this message will be published, as the first one disappeared immediately after pushing "Reply" button.