Titration of standard sabin virus to test sensitivity of cell lines

1.     Scope

·       This procedure explains how to check sensitivity of cells by Titration of Standard lab. Sabin viruses (P1, P2 & P3)

2.     Definition

·       ml        =         Milliliter

·       ART    =         Aerosol Resistant Tips.

·       CCID₅₀ =         Cell Culture Infectious Dose

3.    Material

·       Vortex mixture

·       Microtitre Plate

·       Micropipettes (1000µl & 100µl)

·       Maintenance medium

·       Standard Sabin viruses P1, P2, P3

·       Pipetters with ART tips

·       Flask having confluent monolayer of cells of the cell line to be tested

·       5ml externally threaded sterile vials.

·       Rack for holding the vials.

·       Discarding Pan having 1% hypochlorite solution.

4.     Procedure

·       Cell lines to be tested for sensitivity to all 3 sabin viruses.

·       Keep all the required material in Biosafety cabinet.

·       Arrange 8, 5ml vials in a row in a rack for P1, 8 vials for P2 and 8 vials for P3.

·       Label dilution vials as -2 to -9 for each  serotype

·       Dispense 2.7ml maintenance medium to each vials of 1-8 using 1 ml micropipette.

• First rapidly thaw one aliquot of P1 standard sabin virus and add 0.9ml maintenance medium to make 10^-1 dilution and mix well using vortex mixer.          
• Add 0.3ml of this i.e. 10^-1 dilution to the 1^st vial in the rack labeled as -2 with sterile filter tip to make10^-2 dilution and discard tip into the pan.

·       Close the vial and vortex gently.

10^-3 and discard tip into the pan. Close the vial and vortex gently.

• Repeat dilution steps transferring 0.3ml each time and always changing tip between  dilutions up until vial (10^-9 dilution)
• Label microtitre plate with name of sabin virus, name of cell line to be tested, dilutions, date of titration and cell control wells.
• Add 100µl maintenance medium into wells 12 of rows A to H.
• Add 100µl virus dilution 10^-9 in G1 – G10 and H1 – H1, 10^-8 in E1 – E10 and F1 – F10, 10^-7 in C1 – C10 and D1 – D10, 10^-6 in A1-A10 and B1-B10 i.e. 20 wells for each dilution.
• Repeat the above procedure for P2 and P3 sabin viruses.
• Prepare suspension of cells to be tested (1.5×10⁵/ml for L₂₀B, 1.0X10⁵ /ml for RD cells) calculating at least 12ml per plate.
• Add 100µl of cells from a cell suspension to all wells in rows A to H in the plate with sterile dropper.
• Cover the plate and keep it in the CO₂ incubator at 36ºC and 5% Co₂level with 95% humidity.
• Next day observe the plate for healthy cell monolayer and absence of contamination.
• Read the plate for development of CPE using inverted microscope on 3^rd, 5^th and 7^th day and record readings in Worksheet for Virus Titration PR-44.
• For a valid test cell control should have a complete monolayer of healthy cells and no CPE.
• Lowest dilution should show 100% CPE and highest dilution should not show any CPE and the Intermediate dilutions should show CPE in gradation.
• These plates are to be used for staining.

                            

  Calculate the virus titer using Karber formula.

·       Log CCID₅₀     =          L-d(S-0.5)

·       L                      =          Log of lowest dilution used in the test

·       D                     =          Difference between log dilution steps.

·       S                      =          Sum of proportion of positive test showing CPE.

·       Log CCID₅₀     =          -x

·       Virus Titer      =          10^x CCID₅₀/0.1ml. 

Interpretation

• For initial determination and validation of the titer of the laboratory quality standard, the titer for the quality control standard repeated 3 times and to be parallel each time with the NIBSC reference standard.
• The titers should not vary by more than +/-0.5log10 on all occasions.
• The titer of the lab QC standard will be higher than the NIBSC reference standard.
• If the above criteria are met then the titer for the laboratory QC standard will be the average of the 3 titers.
• Do the titration of the cell lines on 2 occasions i.e. on revival from            LN₂ at #3and #8. Compare virus titer with laboratory QC titer. It must not vary by more than ± 0.5 log of 10 reference value. If there is decline in sensitivity do titration with a fresh aliquot of reference standard to exclude possibility of human error. If titer is again low then discard that cell line and revive a new vial from liquid nitrogen and check its sensitivity again.
• Record the sensitivity of cells in register PR-39.
• Send the results to WHO within 7 days of testing.

1.    Safety conditions

·       To avoid creation of aerosols transfer the fluid gently during dilutions.

·       Vortex the vial, leaves it for one min, and then does the next dilution.

2.    Documentation

·       Virus titration work sheet PR-39 and PR-44.

3.    References used to draw SOP

·       WHO Polio Laboratory Manual.