I'm working on vaccine study
I injected a vaccine candidate and PBS(mock) into mice (2 times with 2-week interval), and collect blood from them to get serum samples.
To test the vaccine efficacy, I performed plaque reduction neutralization test(PRNT) with infectious virus and serial dilution of serum samples.
In the PBS-injected group (negative control), the number of plaques should be equal regardless of the serum concentration. However, in my experiment, the number of plaques decreases as the serum concentration increases in negative control(PBS) group.
Someone told me it could be a matrix effect, but I don't understand why this phenomenon occured.
Why the number of plaques decreases as the serum concentration increases in negative control group?
How can I correct the so called 'matrix effect'?
I just need the equal number of plaques in negative control regardless of serum concentration.
I added a drawing for easy understand of my question.
I would appreciate it if you give me a little help.
Hello Jong, which kind of cells do you use for tritration ? If these cells are non-murine cells and mantained in FCS as usual the problem you describe might be caused by toxicity of mice sera to the cells. If possible, you should include a reference marker for measuring viability or at least have a careful look on the cells whether they are doing fine. Further, it is important to heat inactivate the serum prior to analyses, to avoid falsification of results by complement proteins. Hope this helps, Michael
I agree with comments from Michael. Additionally, you are not seeing a linear decrease in plaque counts at lower dilutions of negative sera. What is the plaque count without any serum or what is your challenge virus dose? Your challenge virus dose seems a little lower. It should be around 30 to 50 PFU to overcome the biological variability. I hope you are inoculating at least duplicate or triplicate wells with each dilution and then taking a Mean to have more confidence in results. Finally, you could calculate 50% reduction in plaques as the end point and will see that negative sera give very low PRNT titers compared to sera from mice injected with a vaccine.
purify the serum from both groups using ammonium sulfate precipitation then dialys in trisbuffer. I have the protocol if you want to try. Or isotype your serum and purify using Sepharose column.
Did you heat inactivate your sera before adding to virus and cells? If not, then complement is still activated and can neutralize virus in the absence of specific antibody.
I think the level of specific antibodies is low, no difference with the control serum. Route of administration ( intraperitoneal is usually effective ), the injected antigen: quality, and quantity amount received by the mouse.