Preparation of authentic sabin virus reference standard from NIBSC strain

1.     Scope

·       This procedure explains how to prepare laboratory control standards from

       authentic sabin virus reference standard received from NIBSC.

2.     Definition

·       ml        =          Milliliter.

·       ART    =          Aerosol Resistant Tips.

·       µl        =          Microlitre

·       CPE    =           Cyto Pathic Effect

3.    Materials required:

·       Reference material:

o   NIBSC – Poliovirus reference sabin type 1 (01/528).

o   NIBSC-   Poliovirus reference sabin type 2 (01/530).

o   NIBSC – Poliovirus reference sabin type 3 (01/532).

·       Characterization: - Standard sabin virus P1, P2, & P3.

·       Handling: - Handle by Jignaben and Ameeben

·       Receiving: - Date of received entered in stock register.

·       Storage : - 20⁰C

·       Use: - These are used in preparation of laboratory quality control standards mentioned in Sop no.5.2, which is further used for evaluating cell culture sensitivity by titration of these standards prepared.

·       Ampouls of each sabin virus P1, P2, and P3 received from ERC-Bombay on       

      30/01/2003

·       Pippeters with ART Tips.

·       Sterile 1.8ml externally threaded vials.

·       Storage box for keeping distributed vials

·       2 ml & 10 ml sterile pipette

·       50 ml centrifuge tubes

·       75cm² flask (containing 30 ml growth medium) of healthy monolayer of L20B cells- 3 no.s

·       75cm² flask of healthy monolayer of RD cells- 3 no.s

1.     Procedure

·       Examine the cells for entire monolayer of healthy cells and absence of contamination by visual examination. A suitable monolayer for use would be formed within two days of seeding.

·       Label the flask to be inoculated (passage no, cell line, date of preparation & virus type).

·       Work with only one serotype at a time in Biosafety cabinet.

·       Inoculate 0.4ml (3^rd ampoule of 0.8 ml received from ERC) of Sabin Polio Reference Standard Virus in 75cm² flask of RD cells.

·       For inoculation of L20B flask retain other half material of ampoule at a later time at -20⁰C.

·       Incubate the inoculated flask at 36⁰C.

·       Examine daily for CPE.

·       When 75% to 100 % CPE appears freeze the flask at -20⁰C and then thaw it.

·       Repeat freezing and thawing for additional two times.

·       Using sterile disposable 10ml pipette, mix and transfer the contents to a labeled 50ml centrifuge tube. After balancing, centrifuge it at 1000 rpm for 10 min.

·       Label each storage vial with the name of the cell line, the name of the virus and date of preparation. 

·       Transfer 0.1ml supernatant to a labeled storage vial.

·       Prepare 250 vials.

·       Store this vials at -20⁰C in deep freeze NO.10, which should be used subsequently as laboratory quality control standard.

·       Repeat the above steps for L20B cells also.

2.    Safety conditions

·       Work only one serotype at a time in biosafety cabinet.

·       To avoid any leakage tighten the centrifuge tubes properly.

·       Before centrifugation check the balance properly.

·       Distribute .0.1ml sabin virus in 1.8ml sterile externally threaded vials gently

3.    Documentation

·       NIBSC & Lab standard Sabin virus register PR-40

4.    References used to draw SOP