STANDARD OPERATING PROCEDURE THEME: BIOTECHNOLOGY (S.O.P.) SECTION: HUMORAL IMMUNOLOGY NUMBER: REVIEW DATE: TITLE: African swine fever virus staining using Immunoperoxidase Monolayer Assay (IPMA) Effective Date: DECEMBER 2021 Supersedes: Written By: ANNA LACASTA-MARIN Date: 12/12/2021 Number Allocated By: Date: Reviewed By: Date: Approved By: Date: 1.0 PURPOSE To describe the Immunoperoxidase Monolayer Assay to detect ASFV infection in WSL cells. 2.0 SCOPE This protocol describes the Immunoperoxidase Monolayer Assay to detect African swine fever virus (ASFV) infection in WSL cells. 3.0 RESPONSIBILITIES It is the responsibility of the technicians and scientists to ensure that the procedure is followed. 4.0 PROCEDURE IPMA is a six-step procedure: 1) Culture the WSL cells 2) Infect the cells with ASFV 3) Incubate the infected cells for 5 days 4) Virus detection with rabbit anti-ASFV 5) Development of the reaction 6) Reading the plates IPMA can be performed in any multi-well culture plate size (6-well, 24-well, 49-well, 96-well), the number of cells and the quantity of virus used will need to be adjusted. In the present protocol, 96-well plates will be the choice for IPMA. Cells are cultures at day -1 in a cell culture incubator to get a confluence of 80% the day after. The following day cells are infected at an MOI of 1 (1 virus: 1 cell) and incubated for 5 days at 37 °C 5% CO2 in a cell culture incubator. After fixing the cells with absolute ethanol for 20 minutes at -20 °C, the endogenous peroxidase is inhibited using hydrogen peroxide for 15 minutes at room temperature in the darkness. Cells are then washed with 0.05%PBS-Tween20 and the ASFV immune sera is added. After 1h incubation at 37 °C the cells are washed, and the secondary TITLE: African swine fever virus staining using Immunoperoxidase Monolayer Assay (IPMA) antibody added (anti-pig: HRP) and incubated for an extra 1 h at 37 °C. The reaction is developed with AEC reagents and plates are observed under the microscope to identify the infected cells. This protocol should be followed carefully to ensure reproducibility. MATERIALS Item name Brand Catalogue number 96-well plate flat bottom Corning 3598 RPMI 1640 medium powder Merk R4130-10L Foetal Calf Serum Gibco 10270106 L-Glutamine Carl-Roth HN08.2 Penicillin/Streptomycin Carl-Roth HP10.1 Gentamycin Carl-Roth HN09.2 b-mercapthethanol Merk 4227.3 Absolute ethanol Merk E7023-500ML Methanol-H2O2 Merk H1009 Anti-pig: HRP Sigma A5670-1ML AEC substrate buffer Merk 152226-50ML AEC reagent Merk 152224-10ML Phosphate buffered saline (PBS) Merk P3813-10PAK Tween 20 Merk 822184 Anti-ASFV, anti-p30 and anti-p54 N/A (In-house reagent) N/A (In-house reagent) from IACUC2021-05 Anti-rabbit: HRP Invitrogen 65-6120 1. Culture media complete RPMI (cRPMI) Reagent Volume (ml) RPMI 1640 cell culture medium 439 foetal bovine serum heat inactivated 50 L-Glutamine 200 mM 5 Penicillin/Streptomycin 100x 5 2-Mercapto-ethanol (Stock at 5x10-2 M ) 0.5 Gentamycin (50mg/ml) 0.5 Store at 4 °C. Use within 6 months. Page 2 of 5 TITLE: African swine fever virus staining using Immunoperoxidase Monolayer Assay (IPMA) 2. Washing Buffer (WB) (PBS-Tween 20) Materials KH2PO4, Na2HPO4, NaCl, Tween 20, deioinsed water Method Combine the following: KH2PO4 2.4 g Na2HPO4 13.8 g NaCl 96 g Tween 20 12 ml Deionized water to 12 litres The pH should be between 7.2 and 7.4. If the pH is not within this range, then it can be adjusted with HCl or NaOH. Store at room temperature. Use within 6 months. METHOD Work inside the biosafety cabinet for all the steps, we need to keep all the reagents sterile. Only the washes can be done outside the biosafety cabinet. DAY -1 (A) Seed the cells into 96-well plates Cells Number of cells Cell culture media 5 1% RPMI, 1% L-Glutamine, 1% Pen/Strept, 10% PAMS 5x10 cells/well FBS 4 1% DMEM, 1% L-Glutamine, 1% Pen/Strept, 10% WSL 2x10 cells/well FBS DAY 0 (B) Virus preparation ASFV1033-GFP-A238LKO. Dilute the virus to get an moi of 1 depending on the type of cell to infect. See table below: Cells Number of virus Cell culture media 5 1% RPMI, 1% L-Glutamin, 1% Pen/Strept, 10% PAMS 5 x10 cells/well FBS 4 1% DMEM, 1% L-Glutamin, 1% Pen/Strept, 5% WSL 4 x10 cells/well FBS Page 3 of 5 TITLE: African swine fever virus staining using Immunoperoxidase Monolayer Assay (IPMA) (C) Virus infection. 1) Discard the media form the cells. 2) Dispense 100 µl/well of the pre-diluted virus. For virus titration use at least 6 wells form the plate. 3) Incubate for 5 days at 37 °C 5% CO2 in a cell culture incubator. DAY 5 (D) Immunoperoxidase Monolayer Assay (IPMA) 1) Fix the plates with absolute ethanol (100 µl/well) and put them at -20 °C for at least 20 minutes. Plates can be stored with absolute ethanol at -20 °C for weeks 2) Take out the plates from -20 °C, gently discard the ethanol by flicking the plate and then blotting once on clean paper. 3) Inhibition of endogenous peroxidase by hydrogen peroxide (without previous washing step): add 100 µl/well of methanol-H2O2 solution. Incubate 15 minutes at RT in darkness. 4) Wash the plate with 150 µl/well of PBS-T. Discard the solution by flicking the plate and then blotting once on clean paper. Repeat washing step for a total of 3 times. 5) Add 150 µl/well of ASFV immune sera (diluted 1:200 in PBS-T). Incubate 1 hour at 37 °C. ASFV immune sera: anti-ASFV, anti-p30 or anti-p54 from IACUC2021-05. 6) Wash the plate with 150 µl/well of PBS-T. Discard the solution by flicking the plate and then blotting once on clean paper. Repeat washing step for a total of 3 times. 7) Add 50 l/well of Protein A-Peroxidase (diluted 1:750 in PBS-T). Incubate 1 hour at 37 °C. 8) Wash the plate with 150 µl/well of PBS-T. Discard the solution by flicking the plate and then blotting once on clean paper. Repeat washing step for a total of 3 times. 9) Add 50 l/well of AEC working solution (see table 2). Incubate 10 minutes in darkness. 10) Wash the plate with 100 µl/well of PBS-T. Discard the solution by flicking the plate and then blotting once on clean paper. Repeat washing step for a total of 2 times. 11) Add 100 µl PBS/well to analyse. Keep the plate covered with aluminium foil and check under the microscope. Page 4 of 5 TITLE: African swine fever virus staining using Immunoperoxidase Monolayer Assay (IPMA) Table 2. Preparation of AEC working solution AEC working solution 300l Stock AEC solution (4 °C) 5 ml Acetate Buffer 3l H2O2 5.0 DEFINITIONS PBS: Phosphate-Buffered Saline PBS-T20: PBS 1X pH 7.4 with 0,1% Tween20. RT: room temperature 6.0 REFERENCES None. 7.0 ATTACHMENTS REVISION HISTORY Revision No. Supercedes Reason This document has a Creative Commons Attribution 4.0 International Licence Funding for this work was provided by the CGIAR Research Program on Livestock Recommended citation: Lacasta, A. 2021. African swine fever virus staining using Immunoperoxidase Monolayer Assay (IPMA). Standard Operating Procedure. Nairobi, Kenya: ILRI. Standard Operating Procedure. Nairobi, Kenya: ILRI. Page 5 of 5