INDIRECT IMMUNOFLUORESCENT ANTIBODY (IFA) STAINING (Appendix IV)

 

FOR VIRAL CULTURE CONFIRMATION

I. Introduction

The IFA technique is used to identify viral isolates in the cells obtained from shell vials and tube cultures. The indirect method consists of two steps. In the first step, primary antibodies are allowed to react with viral antigens in the cells. These specific complexes are detected in a second step using a species-specific antibody conjugated with a fluorochrome. Viruses which we currently identify by IFA staining include cytomegalovirus immediate early antigen (CMV-IE) and enteroviruses.

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II. Reagents and Materials

Virus-specific antibody

FITC-conjugated antimouse antibody

Phosphate buffered saline (PBS)

Distilled water

Cold acetone (4^oC)

Mounting fluid

Sterile pipettes

Cytospin and accessories (for tube cultures)

Humidified chamber

Sterile freezer vial

Glass slides

Coverslips

Paper towels for blotting

Humidified chamber (for tube culture)

 

III. Procedure

1. Shell Vial

Follow outline in Appendix II to determine if staining should be done in the shell vial itself or if a cytospin needs to be prepared. If the staining is to be done in the shell vial itself, proceed to step i) below.

i. Discard cap. Remove maintenance medium from the shell vial using a clean sterile pipette.

a) Add 1 mL of cold acetone. Cover and fix for 10 minutes.

b) Decant acetone and blot on paper towel.

c) Gently rinse with PBS from squirt bottle, filling vial 3/4 full. Decant PBS.

d) Add 75 ml (2 drops from bottle) of appropriate antibody. Cover.

vi. Incubate at 36^oC for 30 minutes.

vii. Gently rinse with PBS from squirt bottle, filling vial 3/4 full. Decant PBS. Repeat.

viii. Add 75ml (2 drops from bottle) of appropriate FITC -conjugated antibodies, cover and repeat steps vi and vii.

x. Remove the coverslip and place cell side down onto a drop of mounting fluid on a glass slide.

xi. Read using fluorescence microscope with the FITC/Evans Blue filter and the 40x objective.

 

2. Tube Culture (or Shell Vials for CPE)

i. Prepare cytospin preparation from cell culture as outlined in Appendix XX.

ii. Add 20 ml of appropriate antibodies onto the fixed cytospin slide.

iii. Incubate in a humidified chamber for 30 minutes at 36^oC.

 

iv. Wash each slide 3 times with fresh PBS for 2 minutes each in a coplin jar.

v. Wipe excess PBS from the slide without touching the cell spot.

vi. Add 20 ml of appropriate FITC-conjugated antibodies.

vii. Incubate in a humidified chamber at 36^oC for 30 minutes.

viii. Wash each slide 3 times with fresh PBS for 2 minutes each in a coplin jar.

ix. Wash with distilled water for 1 minute in a coplin jar.

x. Wipe excess water from the slide without touching the cell spot.

xi. Mount using coverslip and mounting fluid.

xii. Read with fluorescence microscope with the FITC/Evans Blue filter and the 40x objective.

 

Interpretation of Results

Positive: Enterovirus:

An green fluorescence.

Negative: Red cells with no apple-green fluorescence.

 

IV. Quality Control

Appropriate positive and negative control slides should be stained with each batch.

 

V. Reporting

See individual specimen protocols.

 

VI. Reference

Isenberg, H.D., 1992. Clinical Microbiology Procedures Handbook Vol. 2. ASM Press.

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DIRECT IMMUNOFLUORESCENT ANTIBODY (DFA) STAINING (Appendix V )

I. Introduction

The DFA staining technique is used to detect viruses either directly in patient specimens or which have been isolated in shell vial or tube cultures. The method consists of a single staining step using a virus-specific antibody which is conjugated with a fluorochrome. Viruses which we currently identify by DFA staining include HSV-1, HSV-2, VZV, CMV (late antigen) and respiratory viruses (SimulFluor stains for respiratory syncytial virus, parainfluenza, influenza, adenovirus).

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II. Reagents and Materials

FITC-conjugated virus-specific antibody

FITC/Rodamine-conjugated virus-specific antibody (SimulFluor)

Phosphate Buffered Saline (PBS)

dH₂O

cold acetone (4^oC)

mounting fluid

sterile pipettes

cytospin and accessories (for tube culture)

humidified chamber

glass slides

coverslips

paper towels for blotting

 

III. Procedure

 

1. Shell Vial

This procedure is for staining of cells directly in shell vial. If staining a cytospin slide or slide made directly from a patient specimen, follow the tube culture procedure below.

i. Discard cap. Remove maintenance medium from the shell vial using sterile pipette.

ii. Add 1 mL of cold acetone. Cover with tray lid and let sit for 10 minutes.

iii. Decant acetone and blot shell vial on paper towel.

iv. Gently rinse with PBS from squirt bottle, filling vial 3/4 full. Decant PBS.

v. Add 75ml (2 drops from bottle) of appropriate FITC or Rodamine-conjugated virus-specific antibody. Cover with tray lid.

vi. Incubate at 36^oC for 30 minutes.

vii. Gently rinse with PBS from squirt bottle, filling vial 3/4 full. Decant PBS. Repeat.

viii. Remove the coverslip from each shell vial and place cell side down onto a drop of mounting fluid on a glass slide.

ix. For HSV 1, HSV 2, VZ and CMV, read using fluorescence microscope with the FITC/Evans Blue filter and the 40x objective.

x. For respiratory viruses, read using fluorescence microscope with the FITC/Evans Blue Rodamine bi-filter and the 40x objective.

 

2. Tube Culture

i. Prepare cytospin slide from cell culture tube as outlined in Appendix XX.

ii. Fix slide in cold acetone for 10 minutes in a coplin jar. Remove slide and air dry.

iii. Add 20ml of appropriate FITC or Rodamine -conjugated antibody onto the fixed cytospin slide.

iii. Incubate in a humidified chamber at 36^oC for 30 minutes.

iv. Wash each slide 3 times with fresh PBS for 2 minutes each in a coplin jar.

v. Wash with distilled water for 1 minute in a coplin jar.

vi. Wipe excess water from the slide without touching the cytospin preparation.

vii. Mount using coverslip and mounting fluid.

xi. For HSV 1, HSV 2, VZ and CMV, read using fluorescence microscope with the FITC/Evans Blue filter and the 40x objective.

viii. For respiratory viruses, read using fluorescence microscope with the FITC/Evans Blue Rodamine bi-filter.

 

Interpretation of Results

Positive: Bartel CMV monoclonal antibody: Bright apple green fluorescence of cytoplasmic inclusion (late antigen) and homogenous early nuclear antigen in CMV-CPE cells.

Chemicon SimulFluor Respiratory Screen:

All respiratory viruses except RSV show bright apple green fluorescence of the cytoplasm and/or nucleus of the infected cell.

RSV shows bright gold fluorescence of the cytoplasm and/or nucleus of the infected cell.

Chemicon SimulFluor Flu A/Flu B:

Influenzae A virus shows bright apple green fluorescence.

Influenzae B virus shows bright gold fluorescence.

Chemicon SimulFluor RSV/Para 3:

RSV virus shows bright apple green fluorescence.

Parainfluenzae 3 shows bright gold fluorescence.

Chemicon SimulFluor Para 123/Adeno:

Parainfluenza 1,2,3 viruses show bright apple green fluorescence.

Adenovirus shows bright gold fluorescence.

Chemicon individual monoclonal antibodies:

Parainfluenzae 1 and 2, and adenovirus show bright apple green fluorescence.

Negative: Red Cells with no apple-green fluorescence.

 

IV. Quality Control

Appropriate positive and negative control slides should be stained with each batch.

 

VI. Reference

Isenberg, H.D., 1992, ASM. Clinical Microbiology Procedures Handbook Vol. 2.

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