UNCONTROLLED COPY

SAM 315.03

Page 1 of 8

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 315

Supplemental Assay Method for the Detection of Serum Neutralizing 

Antibodies to Rabies Virus using the Rapid Fluorescent Focus Inhibition Test 

(RFFIT)

Date: November 13, 2014 Approved, Pending Standard Requirement

Number: SAM 315.03

Supersedes: SAM 315.02, February 11, 2011

Standard Requirement:

Contact: Alethea M. Fry, (515) 337-7200

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 09Mar15

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 23Mar15

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 23Mar15

Rebecca L.W. Hyde, Section Leader

Quality Management

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
Center for Veterinary Biologics SAM 315.03

Testing Protocol Page 2 of 8

Supplemental Assay Method for the Detection of Serum Neutralizing Antibodies to Rabies Virus using the 

Rapid Fluorescent Focus Inhibition Test (RFFIT)

UNCONTROLLED COPY

Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Cell culture

2.3 Growth medium and diluent

2.4 Virus

2.5 Serum controls

2.6 Other materials

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the sample

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions
Center for Veterinary Biologics SAM 315.03

Testing Protocol Page 3 of 8

Supplemental Assay Method for the Detection of Serum Neutralizing Antibodies to Rabies Virus using the 

Rapid Fluorescent Focus Inhibition Test (RFFIT)

UNCONTROLLED COPY

1. Introduction

This Supplemental Assay Method (SAM) describes the Rapid Fluorescent Focus Inhibition Test 

(RFFIT) which is a serum neutralization method for assaying rabies antibody titers of animals 

used in rabies immunogenicity tests. The RFFIT utilizes fivefold dilutions of serum and a 

constant amount of virus inoculated onto baby hamster kidney cells (BHK21). The cells are 

incubated 20 to 24 hours and stained by the fluorescent antibody technique. The titer of a serum 

is the dilution at which 50% of the examined microscopic fields contain at least 1 fluorescing 

cell.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Vortex mixer (Vortex-2 Genie, Model G-560, Scientific Industries Inc.)

2.1.2 Cabinet, laboratory biosafety level-2 

2.1.3 Water bath, 56°± 2°C 

2.1.4 Micropipettor, 200-μL and 1000-μL single channel, 5- to 50-μL x 12-

channel and tips 

2.1.5 Centrifuge and rotor (Model J6-B centrifuge and Model JS-4.0 rotor), 

Beckman Instruments Inc.

2.1.6 Microscope (fluorescent)

2.2 Cell culture

A baby hamster kidney suspension cell line designated BHK21, 13-S is used.

2.3 Growth medium and diluent

Glasgow minimum essential medium supplemented with 10% tryptose phosphate broth 

and 10% fetal bovine serum (pH 7.0-7.1) is used to grow cells. The same medium 

without fetal bovine serum is used as diluent for serum and virus.
Center for Veterinary Biologics SAM 315.03

Testing Protocol Page 4 of 8

Supplemental Assay Method for the Detection of Serum Neutralizing Antibodies to Rabies Virus using the 

Rapid Fluorescent Focus Inhibition Test (RFFIT)

UNCONTROLLED COPY

2.3.1 Glasgow minimum essential medium (MEM) 20013

1. 12.5 g Glasgow MEM

2. 2.75 g sodium bicarbonate (NA2HCO2)

3. 2.0 g tryptose phosphate broth

4. Q.S. to 1000 mL with deionized water (DI).

5. Adjust pH to 6.8

6. Sterilize through a 0.22-μm filter.

7. Store at 2°- 7°C.

2.3.2 Diethylaminoethyl (DEAE) dextran (1%) (National Centers for Animal 

Health (NCAH) Media #30163)

1. 1.0 g DEAE dextran

2. Q.S. to 100 mL with DI.

3. Sterilize through a 0.22-μm filter

2.3.3 Heat-inactivated, rabies antibody-free horse serum (10 mL)

2.3.4 Kaolin (25%)

1. 25 g kaolin

2. Q.S. to 100 mL with DI.

3. Sterilize through a 0.22-μm filter

2.4 Virus

CVS-11 strain of rabies virus.

2.5 Serum controls

A negative reference serum and a low positive reference serum are assayed in each test.
Center for Veterinary Biologics SAM 315.03

Testing Protocol Page 5 of 8

Supplemental Assay Method for the Detection of Serum Neutralizing Antibodies to Rabies Virus using the 

Rapid Fluorescent Focus Inhibition Test (RFFIT)

UNCONTROLLED COPY

2.6 Other materials

2.6.1 Polystyrene tubes 12 x 75-mm

2.6.2 1% diethylaminoethyl (DEAE) dextran in growth media

2.6.3 Rabies conjugate, current lot

2.6.4 Plastic 8-chamber tissue culture slides

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have experience in laboratory dilution techniques and the handling and 

discarding of human pathogens,

CAUTION: Live rabies is a potential deadly human pathogen! To work with live 

rabies, personnel must be vaccinated for rabies and monitored for a minimum 

rabies titer acceptable to appropriate human health officials. The use of 

appropriate biological safety cabinets is required for dilution of challenge material. All 

discarded challenge materials should be considered potentially infective and handled in a 

manner consistent with safe laboratory practices and in accordance with the 

recommendations of the Centers for Disease Control and Prevention (CDC) and the

National Institutes of Health (NIH). 

3.2 Preparation of equipment/instrumentation

On the day of test initiation, set a water bath at 56°± 2°C.

3.3 Preparation of reagents/control procedures

Serum to be tested is heat-inactivated at 56°C for 30 minutes. Equal volumes of serum 

and 25% kaolin are mixed and allowed to stand at room temperature for 20 minutes with 

frequent agitation. The mixture is then centrifuged at 1500 x g for 15 minutes to remove 

the kaolin. Treated serum is considered to be diluted 1:2.

3.4 Preparation of the sample

Each serum is diluted in the chambers of the plastic slide (4 chambers/serum). A 0.05 mL 

volume of diluent (Glasgow medium with 10% tryptose phosphate broth and without 

fetal bovine serum) is dispensed into the first chamber and 0.2 mL is dispensed into the 

next 3 chambers. An initial 1:2.5 dilution is made by adding 0.2 mL of treated serum 
Center for Veterinary Biologics SAM 315.03

Testing Protocol Page 6 of 8

Supplemental Assay Method for the Detection of Serum Neutralizing Antibodies to Rabies Virus using the 

Rapid Fluorescent Focus Inhibition Test (RFFIT)

UNCONTROLLED COPY

(considered a 1:2 dilution) to the 1st chamber. The contents of the chamber are mixed, 

0.05 mL is transferred to the next chamber to make a fivefold dilution, and 0.1 mL from 

the 1st chamber is discarded. The fivefold dilution process is continued for the next 2 

chambers with 0.1 mL being discarded from the 2nd and 3rd chambers and 0.15 mL 

being discarded from the 2nd and 3rd chambers and 0.15 mL being discarded from the 

4th chamber. The final result is 4 fivefold dilutions (1:2.5, 1:12.5, 1:62.5, and 1:312.5) of 

0.1 mL in each chamber.

4. Performance of the Test

4.1 Preparation and addition of virus

The CVS-11 strain of rabies virus is diluted to contain 30-100 fluorescing foci 

dose50/0.1 mL. A fluorescing foci dose50 (FFD50) is the amount of virus necessary to 

produce at least 1 fluorescing cell in 50% of the microscopic fields examined. A 0.1 mL 

volume of diluted virus is added to each chamber. This addition results in final serum 

dilutions of 1:5 through 1:625. A backtitration (tenfold dilutions) of the indicator virus is 

performed in tubes and 0.1 mL of the working dilution, the 10-1dilution, and the 10-2

dilution are added to chambers containing 0.1 mL of diluent. The backtitration and the 

virus-serum mixture are incubated in a 5% CO2 incubator at 37°C for 90 minutes.

4.2 Preparation and addition of cells

Two- to 4-day-old monolayers of BHK21, 13-S cells are split approximately 30 minutes 

prior to completion of the virus-serum mixture incubation period. The cells are 

standardized to 8 x 105cells/mL in growth medium containing 10 μg/mL of DEAEdextran. The cells are allowed to stand at room temperature for at least 10 minutes. At the 

conclusion of the 90 minute incubation period, the treated cells are dispensed onto the 

slides, 0.2 mL per chamber. The slides are then incubated at 37°C in a 5% CO2 incubator 

for 20 to 24 hours.

4.3 Staining procedure

The plastic chambers are removed from the slide in such a way that the gasket is left 

attached to the slide. The slides are gently dipped in Dulbecco’s phosphate buffered 

saline (PBS), fixed in cold (oC) acetone for 10 minutes and dried. Rabies conjugate is 

diluted in PBS and dispensed onto each slide. The slides are incubated at 37°C for 30 

minutes, placed in a PBS bath for 10 minutes, rinsed in deionized water, and dried.
Center for Veterinary Biologics SAM 315.03

Testing Protocol Page 7 of 8

Supplemental Assay Method for the Detection of Serum Neutralizing Antibodies to Rabies Virus using the 

Rapid Fluorescent Focus Inhibition Test (RFFIT)

UNCONTROLLED COPY

5. Interpretation of the Test Results

Twenty microscopic fields at 130X are examined in each chamber and the number of 

microscopic fields containing at least 1 cell with specific rabies fluorescence is recorded. The 

virus neutralizing titer of a serum is the final dilution of serum in which 10 to 20 fields contain at 

least 1 fluorescing cell. The serum titer is calculated by the method of Spearman-Kärber.

The results of the serum controls and the virus backtitration are evaluated to determine the 

validity of the test. At least 15 of 20 fields of the 1:5 dilution of the negative reference should 

contain fluorescing cells and the titer of the positive control reference should be within 2.5-fold 

of its established geometric mean. The backtitration of the indicator virus should be within the 

following ranges:

Undiluted - 20 of 20 fields positive

10-1dilution - ≥ 18 of 20 fields positive

10-2dilution - ≤ 10 of 20 fields positive

6. Report of Test Results

The virus neutralizing titer of a serum is the final dilution of serum in which 10 to 20 fields 

contain at least 1 fluorescing cell. 

7. References

Smith JS, Yager, PA, Baer, GM. A rapid fluorescent focus inhibition test (RFFIT) for 

determining rabies virus neutralizing antibody. In Laboratory Techniques in Rabies, 4th ed., 

Meslin, FX, MM Kaplan, and H Koprowski, eds. World Health Organization, Geneva. 1996, 

Chpt 15.

8. Summary of Revisions

Version .03

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.

Version .02 (February 11, 2011)

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 
Center for Veterinary Biologics SAM 315.03

Testing Protocol Page 8 of 8

Supplemental Assay Method for the Detection of Serum Neutralizing Antibodies to Rabies Virus using the 

Rapid Fluorescent Focus Inhibition Test (RFFIT)

UNCONTROLLED COPY

the outcome of the test, the following changes were made to the document:

 The Contact information has been updated.

 2.1: A list of test equipment has been added.

 2.2: Media formulations have been included in the document.

 3.1: Training requirements and associated hazards have been added.

 3.2: Criteria for water bath temperature have been added. 

 6: Criteria for test reporting have been added

 7: Reference information has been added.