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SAM 322.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 322

Supplemental Assay Method for Determination of the Specific Viral Antigen 

Content in Inactivated Canine Coronavirus Vaccines

Date: January 9, 2015 Approved, Pending Standard Requirement

Number: SAM 322.05

Supersedes: SAM 322.04, 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 Determination of the Specific Viral Antigen Content 

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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 Determination of the Specific Viral Antigen Content 

in Inactivated Canine Coronavirus Vaccines

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1. Introduction

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

(ELISA) method for the quantitation of the viral antigen content of inactivated canine 

coronavirus (CCV) vaccines. The relative potency (RP) of CCV vaccines is determined by 

comparing the amount of CCV in a Test Vaccine to the CCV content of a Reference Preparation 

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

study.

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 micropipettes with tips of appropriate size 

2.1.5 Microtiter plate shaker (Model 4625, Labline Instruments Inc.)

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

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

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

2.1.8 Vortemp Shaker 

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. 1.9 g sodium phosphate, dibasic, anhydrous (Na2HPO4)
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2. 0.22 g sodium phosphate, monobasic, monohydrate (NaH2PO4•H2O)

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 121°± 2°C, 15 psi 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 2 N HCl.

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

2.2.3 Blocking Solution

1. 1 g casein in 100 mL Carbonate Coating Buffer

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

2.2.4 Wash Buffer

1. 500 μL Tween-20 in 1000 mL 0.01 M PBS

2. Store at room temperature.

2.2.5 Diluent Buffer

1. 1 g casein in 100 mL Wash Buffer

2. Store at 2°- 7°C; use within 1 week.
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2.2.6 Feline infectious peritonitis virus antibody (FIPV Ab), ammonium sulfateprecipitated

2.2.7 CCV monoclonal antibody (CCV MAb)

2.2.8 Rabbit anti-mouse horseradish peroxidase conjugate (Rabbit 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 Flat bottom, 96-well ELISA plate (ELISA Plate)

2.2.11 Plate sealer

2.2.12 Reference Preparation. Each manufacturer provides a Reference 

Preparation 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 

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

in the APHIS filed Outline of Production.

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

The microplate reader must be turned on at least 30 minutes prior to determination of OD 

readings. The microplate reader is zeroed on air prior to initial use.
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3.3 Preparation of reagents/control procedures

3.3.1 Test Plate preparation. On the day of plate coating, dilute the FIPV Ab, 

per the CVB Reagent Data Sheet, in Carbonate/Bicarbonate Coating Buffer. Mix 

by vortexing and pipette 100 μL of diluted FIPV Ab to each well of an ELISA 

Plate, which becomes the Test Plate. Cover the Test Plate with a plate sealer and 

incubate 3 ± 2 days, at 2°- 7°C.

3.3.2 CCV MAb preparation: On the day the Test Plate is read, dilute the CCV 

MAb, per the CVB Reagent Data Sheet, in Diluent Buffer; mix by vortexing.

3.3.3 Rabbit Anti-Mouse Conjugate preparation: On the day the Test Plate is 

read, dilute the Rabbit Anti-mouse Conjugate, per previously determined optimal 

dilution, in Diluent Buffer; mix by vortexing.

3.3.4 Substrate Solution preparation. On the day the Test Plate is read, just prior 

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

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

Solution must remain clear. The Substrate Solution must 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 an 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 or special outline. 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 reference, the extraction procedure is not 

required for the Reference Preparation. 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.

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). Twofold 

dilutions may be made in an additional ELISA Plate, which becomes the Transfer 

Plate, as follows (tips are changed between each dilution):

1. Add 150 μL of Diluent Buffer to wells in rows B-H with a 12-channel 

micropipettor (see Appendix).

2. The starting dilution for the Reference Preparation and the Test 
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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.

3. Add 300 μL of the starting dilution of the Reference Preparation to 

wells A1 and A2.

4. Add 300 μL of the starting dilution of the Test Vaccine to wells A3 

and A4. Additional Test Vaccines may be tested in duplicate in columns 

5-12.

5. Transfer 150 μL from row A to row B. Mix row B with the 12-channel 

micropipettor (7 ± 2 fills).

6. Continue as in Step 5 for the remaining rows C-G, transferring 150 μL

from the previous row to the next row. 

Note: Row H is not used and remains as Diluent Buffer for transfer to 

the blank wells.

4. Performance of the Test

4.1 Remove the Test Plate prepared earlier from 2°- 7°C storage (see Section 3.3.4).

4.2 Decant the Test Plate contents in a suitable container. Fill each well with at least 

200 μL of Wash Buffer. Immediately decant Wash Buffer from the Test Plate. Repeat for 

a total of 4 washes. At no time should wells dry between rinses or incubations. After the 

last wash, tap the Test Plate on paper towels to remove residual Wash Buffer. An 

automatic plate washer may be used.

4.3 Add 200 μL of Blocking Solution to each well of the Test Plate; seal the Test 

Plate with a plate sealer and incubate for 60 ± 10 minutes at 36°± 2°C.

4.4 Wash the Test Plate as in Section 4.2.

4.5 Transfer 100 μL of the Test Vaccine and the Reference Preparation diluted on the 

Transfer Plate (see Section 3.4.2) to corresponding wells of the blocked Test Plate. Tips 

need not be changed if proceeding from the most dilute to the most concentrated (Row H 

to Row A). Row H receives all assay reagents except antigen and serves as the blank 

wells. 

4.6 Seal the Test Plate with a plate sealer and incubate on the microtiter plate shaker 2 

hours ± 10 minutes, or overnight (14 ± 2 hours), at 36°± 2°C with sufficient agitation to 
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keep the test samples in suspension. The incubation time shall be stated in Part V of the 

APHIS filed Outline of Production or special outline. If the incubation time is not 

specified, incubation will be overnight.

4.7 Wash the Test Plate as in Section 4.2.

4.8 Pipette 100 μL/well of Diluted CCV MAb to each well of the Test Plate; seal the 

Test Plate with a plate sealer and incubate for 60 ± 10 minutes at 36°± 2°C.

4.9 Wash Test Plate as in Section 4.2.

4.10 Pipette 100 μL of Diluted Rabbit Anti-mouse Conjugate to each well of the Test 

Plate; seal the Test Plate with a plate sealer and incubate for 60 ± 10 minutes at 36°± 2°C.

4.11 Wash the Test Plate as in Section 4.2.

4.12 Pipette 100 μL of Substrate Solution to each well of the Test Plate; incubate the 

Test Plate at room temperature.

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

wavelength on the microplate reader when the color development gives a sufficient OD 

reading in at least the fourth dilution of the Reference Preparation (OD ≥ 0.05 after the 

average blank reading is subtracted).

4.14 Determine the arithmetic mean of at least 3 wells of the blank wells in Row H; 

this becomes the Average Blank Reading. The Average Blank Reading is subtracted from 

all readings before analysis of the data by the current version of RelPot.

4.15 Evaluate the data using RelPot.

5. Interpretation of the Test Results

5.1 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).

5.2 For a 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 

stated in an APHIS filed Outline of Production.

5.3 For a Test Vaccine with an RP value less than the RP stated in an APHIS filed 

Outline of Production, the test may be repeated when the test meets the criteria defined in 

9 CFR 113.8(c)(5) (see current version of RelPot, Test Results and Interpretation).
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6. Report of Test Results

Record RP results on the test record.

7. References

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

Washington, DC.

7.2 Horsburgh BC, Brown TDK. Sequence analysis of CCV and its relationship to 

FIPV, TGEV and PRCV. In: Coronaviruses, Laude H, Vautherot J, eds., pp 3-9. Plenum 

Press, New York, 1994.

7.3 Katz JB, Hanson SK, Patterson PA, Stoll IR. In vitro assessment of viral antigen 

content in inactivated aluminum hydroxide adjuvanted vaccines. J Vir Meth 1989, 

25:101-108.

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.

 2.1.9: A Vortemp shaker has been added to the list of equipment.

Version .02

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 
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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.

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

individual items.

 “Reference and Reagent Sheet” has been changed to “Reagent Data Sheet” 

throughout the document.
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Appendix

Transfer and Test Plate Format

 1 2 3 4 5 6 7 8 9 10 11 12

A REF REF TS1 TS1 TS2 TS2 TS3 TS3 TS4 TS4 TS5 TS5

B

C

D

E

F

G

H BL

K

BL

K

BL

K

BL

K

BL

K

BL

K

BL

K

BL

K

BL

K

BL

K

BL

K

BL

K

REF= Reference Preparation; TS= Test Serial; BLK= Blank