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SAM 321.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 321

Supplemental Assay Method for Quantitating the GP70 Antigen of Feline 

Leukemia Virus in Veterinary Vaccines

Date: January 9, 2015 Approved, Pending Standard Requirement

Number: SAM 321.05

Supersedes: SAM 321.04, June 30, 2011

Standard Requirement:

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

 Sandra K. Conrad

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 12Feb15

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 17Feb15

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 17Feb15

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.
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Supplemental Assay Method for Quantitating the GP70 Antigen of Feline Leukemia 

in Virus Veterinary Vaccines

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

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

Appendix
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Supplemental Assay Method for Quantitating the GP70 Antigen of Feline Leukemia 

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1. Introduction

This Supplemental Assay Method (SAM) describes the enzyme-linked immunosorbent assay 

(ELISA) titration method for quantitation of the 70,000 Dalton glycoprotein (gp70) antigen of 

feline leukemia virus (FeLV) vaccines by the relative potency (RP) method. The RP of a Test 

Vaccine is determined by comparing the amount of gp70 antigen of a Test Vaccine to the gp70 

antigen content of a Reference Preparation that has been directly or indirectly shown to be 

protective in a host animal immunogenicity trial.

2. Materials

2.1 Equipment/instrumentation

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

below.

2.1.1 Incubator 36°± 2°C, 5% ± 1% CO2, high humidity (Model 3158, Forma 

Scientific Inc.)

2.1.2 Microplate reader (Model MRX, Dynex Technologies Inc.)

2.1.3 Microplate washer (Model EL404, Bio-Tek Instruments Inc.)

2.1.4 Single and 12-channel micropipettors and tips of appropriate size. 

2.1.5 Current version of the U.S. Department of Agriculture, Center for 

Veterinary Biologics’ (CVB’s) Relative Potency Calculation Software (RelPot)

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below. All 

reagents and supplies must be sterile.

2.2.1 0.01 M Phosphate buffered saline (PBS)

1. 1.33 g sodium phosphate, dibasic, anhydrous (Na2HPO4)

2. 0.22 g sodium phosphate, monobasic, monohydrate (NaH2PO4•H2O)
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3. 8.5 g sodium chloride (NaCl)

4. Q.S. to 100 mL with distilled water (DW)

5. Adjust pH to 7.2-7.6 with 0.1 N sodium hydroxide (NaOH) or 1.0 N 

hydrochloric acid (HCl).

6. Sterilize by autoclaving at 15 psi, 121°± 2°C for 35 ± 5 minutes.

7. Store at 2°- 7°C.

2.2.2 0.05 M Carbonate/Bicarbonate Coating Buffer, pH 9.6

1. 0.159 g sodium carbonate (Na2CO3)

2. 0.293 g sodium bicarbonate (NaHCO3)

3. Q.S. to 100 mL with DW.

4. Adjust pH to 9.6 with 1.0 N HCl.

5. Store at 2°- 7°C; use within 1 week.

2.2.3 Blotto 

1. 1.5 g nonfat dry milk powder (Flavorite Instant Nonfat Dry Milk, 

extra grade, Preferred Products Inc.) (Brands and lots may vary in 

blocking ability. Determine appropriate percentage of dry milk for each

batch.)

2. 100 mL PBS

3. Add 2 μL of Antifoam A.

4. Store at 2°- 7°C; use within 1 week.

2.2.4 Diluent Buffer 

1. 100 mL Blotto

2. 1.0 mL Triton X-100

3. Store at 2°- 7°C; use within 1 week.
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2.2.5 Washing Buffer 

1. 1000 mL of 0.01 M PBS

2. 1.0 mL Tween-20

3. Store at 2°- 7°C. 

2.2.6 Goat Anti-FeLV gp70 Polyclonal Antibody

2.2.7 Anti-FeLV gp70 monoclonal antibody (MAb)

2.2.8 Goat anti-mouse horseradish peroxidase conjugate (Goat Anti-mouse 

Conjugate) 

2.2.9 (2,2’azino-di-{3 ethyl-benzthiazaline sulfonate 6}) (ABTS) peroxidase 

substrate solution (Substrate Solution)

1. Solution A, ABTS 

2. Solution B, Hydrogen Peroxide

2.2.10 FeLV gp70 Positive Control (available from the CVB)

2.2.11 Reference preparation: Each manufacturer provides a Reference Vaccine 

that has been directly or indirectly shown to be protective in a host animal 

immunogenicity trial. The reference preparation is the lot number identified in 

Part V of the Animal and Plant Health Inspection Service (APHIS) filed Outline 

of Production or special outline. All subsequent serials produced by the 

manufacturer must have an RP equal to or greater than the RP value contained in 

the APHIS filed Outline of Production.

2.2.12 Immulon II®, flat bottom, 96-well plate

2.2.13 Flat bottom, 96-well plate (transfer plate)

2.2.14 Plate Sealer
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3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have training and experience in the immunological basis of antigen 

capture ELISA assays, the principles of Optical Densitometry (OD), and computer 

software analysis.

3.2 Preparation of equipment/instrumentation

On the day of the reading, the ELISA reader must be turned on at least 30 minutes prior 

to determination of the OD. Dual wavelength settings, 405-nm test wavelength measured 

against a 490-nm reference wavelength, are used. The ELISA reader is zeroed on air prior 

to use.

3.3 Preparation of reagents/control procedures

3.3.1 Test plate preparation. One to five days prior to test initiation, dilute the 

Goat Anti-gp70 Polyclonal Antibody, per the CVB Reagent Data Sheet, in 

Carbonate/Bicarbonate Coating Buffer. 

1. Pipette 100 μL/well of the Diluted Goat Anti-FeLV gp70 Polyclonal 

Antibody to all wells of a flat bottom, 96-well Immulon II®plate. This 

becomes the Test Plate.

2. Cover the Test Plate with a Plate Sealer and incubate at 3 ± 2 days, at 

2°- 7°C. 

3.3.2 Diluted gp70 Positive Control preparation. On the day of testing, dilute the 

gp70 Positive Control, per the CVB Reagent Data Sheet, in Diluent Buffer. 

3.3.3 Diluted Anti-gp70 MAb preparation. On the day of testing, dilute the 

Anti-gp70 MAb, per the CVB Reagent Data Sheet, in Blotto.

3.3.4 Diluted Goat Anti-mouse Conjugate. On the day of testing, dilute the Goat 

Anti-mouse Conjugate, per previously determined optimal dilution, in PBS. 

Optimal dilution results in an OD reading between 0.400 and 0.700 when 

incubated at 36°± 2°C for 20 ± 10 minutes.
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3.3.5 Substrate Solution preparation. On the day of testing, just prior to 

substrate addition, mix equal volumes of ABTS Solution A and Hydrogen 

Peroxide Substrate Solution B per the manufacturer’s instructions. The resulting 

Substrate Solution should remain clear and should be at room temperature at time 

of use.

3.4 Preparation of the sample

3.4.1 Antigen Extraction (optional). If the Test Vaccine contains adjuvant which 

interferes with antigen detection, the firm may specify the procedure for 

extraction of the antigen from the adjuvant. If extraction is a necessary step, the 

extraction procedure will be included in Part V of the APHIS filed Outline of 

Production. The CVB will extract antigen using the firm’s protocol. If no protocol

is stated in either the APHIS filed Outline of Production or special outline, the test 

will be conducted at the CVB without extraction. If the Reference Preparation is a 

product reference, both the Reference Preparation and Test Vaccine must be 

treated identically. If the Reference Preparation is a purified preparation, the 

extraction procedure is not required. 

3.4.2 All samples must be at room temperature before testing. The initial test of 

a Test Vaccine will be with a single vial (a single sample from 1 vial). Make 

twofold dilutions of the Reference Preparation and the Test Vaccine in Diluent 

Buffer in a flat bottom, 96-well plate which becomes the Transfer Plate.

3.4.3 An initial dilution of the Reference Vaccine and/or Test Vaccine may be 

made. This dilution is determined by the firm to assure the 6 dilutions tested 

encompass the linear portion of the regression curve. The starting dilution for the 

Reference Vaccine and/or the Test Vaccine shall be stated in Part V of the APHIS 

filed Outline of Production or special outline. Unless stated otherwise, the diluent 

will be Diluent Buffer for the initial dilution. 

1. Pipette 150 μL of Diluent Buffer to wells in rows C-G of the Transfer 

Plate with a 12-channel micropipettor (see Appendix).

2. Pipette 300 μL of the starting dilution of the Reference Preparation to 

3 wells in row B of the Transfer Plate (see Appendix).

3. Pipette 300 μL of the starting dilution of the first Test Vaccine to 3 

adjacent wells in row B of the Transfer Plate. An additional Test Vaccine

may be tested into the next 3 adjacent wells.
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4. With a 12-channel micropipettor and the appropriate number of tips to 

correspond to the number of wells used in a row, mix row B (7 ± 2 fills) 

and transfer 150 μL to row C of the Transfer Plate. Replace tips, mix row 

C, and transfer 150 μL to row D. 

5. Continue as in Step 4 for the remaining rows D-G of the Transfer 

Plate, transferring 150 μL from the previous to the next row.

4. Performance of the Test

4.1 On the day of testing, remove the Plate Sealer and pipette 200 μL of thoroughly 

mixed Blotto to each well of the Test Plate already containing Diluted Goat Anti-gp70 

Polyclonal Antibody. Do not remove the Diluted Goat Anti-gp70 Polyclonal Antibody 

prior to adding the Blotto. Reseal plate and incubate at 2°- 7°C for 90 ± 30 minutes.

4.2 Decant Blotto from the Test Plate. Pipette 200-300 μL of Washing Buffer into 

each well. Immediately decant Washing Buffer from the plate. Repeat for a total of 

3 washes. At no time should wells dry between washes or incubations. An automatic 

microplate washer may be used.

4.3 Transfer 100 μL/well of each dilution in the Transfer Plate (starting in row G, 

most dilute, and ending in row B, least dilute) to appropriate rows of the Test Plate (see 

Appendix). Tips need not be changed between rows when proceeding from most dilute 

to successively more concentrated dilutions. 

4.4 Pipette 100 μL/well of Diluted gp70 Positive Control to wells 11-B, 11-C, and 

11-D of the Test Plate.

4.5 Pipette 100 μL/well of Diluent Buffer to wells 11-E, 11-F, and 11-G of the Test 

Plate to serve as blanks.

4.6 Seal the Test Plate with a Plate Sealer; incubate at 36°± 2°C for 60 ± 10 minutes.

4.7 Wash the Test Plate as in Section 4.2.

4.8 Pipette 100 μl/well of Diluted Anti-gp70 MAb to all wells of the Test Plate.

4.9 Seal the Test Plate with a Plate Sealer; incubate at 36°± 2°C for 60 ± 10 minutes.

4.10 Wash the Test Plate as in Section 4.2.
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4.11 Pipette 100 μL/well of Diluted Goat Anti-mouse. Conjugate to all wells of the 

Test Plate.

4.12 Seal the Test Plate with a Plate Sealer; incubate at 36°± 2°C for 60 ± 10 minutes.

4.13 Wash the Test Plate as in Section 4.2, then wash 2 times with PBS instead of 

Washing Buffer.

4.14 Pipette 100 μL/well of Substrate Solution to all wells of the Test Plate.

4.15 Incubate the Test Plate at room temperature for 20 ± 10 minutes.

4.16 Read the Test Plate at 405-nm test wavelength against a 490-nm reference 

wavelength, when the Positive Control wells (11-B, C, D) read between 0.400 and 0.700 

OD after the average blank reading is subtracted.

4.17 The arithmetic mean of the blank wells is determined; this becomes the average 

blank reading. The average blank reading is subtracted from all readings before analysis 

of the data by RelPot.

4.18 Evaluate results using the current version of RelPot.

5. Interpretation of the Test Results

5.1 If the corrected OD of the Positive Control is not within the 0.400-0.700 OD 

range, the test is considered a NO-TEST and can be repeated without prejudice.

5.2 All validity criteria in the current version of RelPot must be met for a valid test. 

An invalid test may be repeated. Testing may be repeated for EQUIVOCAL tests as 

defined in 9 CFR 113.8(c)(4).

5.3 For a given Test Vaccine to be SATISFACTORY, the RP value of at least 1 

valid RP from the group of the highest scoring valid RP values has to be greater than or 

equal to the RP contained in an APHIS filed Outline of Production. 

5.4 For a Test Vaccine result less than the RP contained in an APHIS filed Outline of 

Production, the test may be repeated if the test meets 9 CFR 113.8(c)(5) criteria.
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6. Report of Test Results

Results are reported as the RP for the Test Vaccine. The highest RP of the top scores shall be 

considered the RP value for reporting purposes.

7. References

7.1 Title 9, Code of Federal Regulations, section 113.8, U.S. Government Printing 

Office, Washington, DC.

7.2 Shibley GP, Tanner JE, Hanna SA, United States Department of Agriculture 

licensing requirements for feline leukemia virus vaccines. JAVMA, (1991), Vol. 199, No. 

10, 1402-1406.

8. Summary of Revisions

Version .05

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

to keep the same next review date for the document.

 Reference to SAM 318 has been removed throughout the document.

Version .04

 The Contact information has been updated.

Version .03

 2.1.4: Changes were made to the type and sizes of micropipettors to allow for 

variation as appropriate.

Version .02

This document was rewritten 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 

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

 The refrigeration temperatures have been changed from 4° ± 2°C to 2°- 7°C. This 

reflects the parameters established and monitored by the Rees system. 

 “Test Serial” has been changed to “Test Vaccine throughout the document.
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 “Reference and Reagent Sheet” has been changed to “Reagent Data Sheet”

throughout the document.

 The footnotes have been deleted with any pertinent references now noted next to the 

individual items.
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Appendix

Transfer and Test Plate Format

Locations for dilution plate:

Row B, columns 2, 3, and 4: lowest dilution of Reference Vaccine Row B, columns 5, 6, and 7: 

lowest dilution of Test Serial 1

Row B, columns 8, 9, and 10: lowest dilution of Test Serial 2

Locations for test plate:

Rows B through G, columns 2, 3, and 4: increasing dilution of the Reference Vaccine

Rows B through G, columns 5, 6, and 7: increasing dilution of Test Serial 1

Rows B through G, columns 8, 9, and 10: increasing dilution of Test Serial 2

B-11, C-11, and D-11: gp70 Positive Control

E-11, F-11, and G-11: Blank

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

R

E

F

E

R

E

N

C

S

E

R

I

A

L

1

S

E

R

I

A

L

1

S

E

R

I

A

L

1

S

E

R

I

A

L

2

S

E

R

I

A

L

2

S

E

R

I

A

L

2

+

C

N

T

R

B

L

A

N

.

E

R

E

F

E

R

E

N

C

E

R

E

F

E

R

E

N

C

E

K

B

C

D

E

F

G

H

A