PCV2 don't cause CPE on PK15 cell line.  You have to do IFA or IPMA.

I agree. Just do a classical TCID50 with anti-capsid primary antibody and an HRP-conjugated secondary antibody. Fast and easy.

If you don't have that, order it or ask a lab that published PCV2 titration.

Good luck.

Hi Nicolaas Van Renne, could you tell more details about how to do that TCID50?

TCID50 is a classic technique in virology first described by Reed and Muench and dates back to the 1930s.

I refer to one of our papers on PCV2 (Huang, Van Renne et al, J Gen Virol 2015 A sequence of basic residues in the porcine circovirus type 2 capsid protein is crucial for its co-expression and co-localization with the replication protein).

In brief: (1) seed PK15 cells in a well plate

(2) take your virus stock and make 1:10 dilutions.

(3) infect half a row of your 96 well plate with the stock, infect the next half row with 1:10 dilution, the third half row with 1:100, etc. 1:1,000,000 will be more than enough. PCV2 stocks generally have only moderate infectivity.

(4) After 72 hrs fix cells, wash, stain with anti-capsid antibody, wash, add HRP-labeled 2nd antibody, wash, add colorizing agent (which will be metabolized by the HRP).

(5) check all wells if they contain positive cells with microscope

(6) calculate the TCID50 according to Reed and Münch.

The TCID50 will give you a measure of infectivity of your stock.

eg. 1.96*10^4 TCID50/mL

Just look for a good tutorial on the internet or a virology textbook, since TCID50 is so basic there will be many good guidelines available.

Good luck

Hi Nicolaas,

Thank you for your protocol. I have several questions about the protocol:

1. Do you use PBS or PBS+tween 80+serum for washing? And do we have to use only horse serum or any serum? I read several protocols that advised to use horse serum.

2. Do you fixate the cells using 0.3% formalin?

3. Do you have suggestions how to manage the antibody dilution? Should we take only 1 uL, dilute it, and re-freeze the rest?

Thank you.