Center for Veterinary Biologics SAM 613.05

Testing Protocol Page 1 of 10

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 613

Supplemental Assay Method for In vitro Potency Testing of 

Erysipelothrix rhusiopathiae Bacterins

Date: June 17, 2022 Approved, pending Standard Requirement 

Number: SAM 613.05 

Supersedes: SAM 613.04, May 18, 2016 

Standard Requirement: 

Contact: Email: Methodsrequest.notification@usda.gov 

Phone: Center for Veterinary Biologics, 515-337-6100 

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 In vitro Potency Testing of Erysipelothrix rhusiopathiae Bacterins

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 

3.4 Preparation of the sample 

4. Performance of Capture ELISA

5. Interpretation of the Test Results

5.1 Relative potency calculation method 

5.2 Requirements for a valid assay 

5.3 Requirements for a satisfactory serial

6. Report of Test Results 

7. Summary of Revisions 

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Supplemental Assay Method for In vitro Potency Testing of Erysipelothrix rhusiopathiae Bacterins

1. Introduction

This Supplemental Assay Method (SAM) for in vitro potency testing of Erysipelothrix 

rhusiopathiae bacterins employs a capture enzyme-linked immunosorbent assay (ELISA) 

specific for the 65-kD protein of E. rhusiopathiae. Relative potency is determined by comparing 

the 

65-kD protein content of the test serial to the 65-kD protein content present in a suitably 

qualified reference bacterin. 

2. Materials

2.1 Equipment/instrumentation 

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

below. 

2.1.1 Microplate reader with dual wavelengths (450 and 650 nm) 

2.1.2 Micropipettors, to cover the range of 5.0-μL to 1000-μL 

2.1.3 8- or 12-channel micropipettor 

2.1.4 Orbital shaker

2.1.5 Balance, to measure 150 mg to 15 gram 

2.1.6 A computer software program capable of implementing the calculations 

described in CVBSOP0102, Using Software to Estimate Relative Potency 

2.1.7 High-intensity ultrasonic processor, 20 kHz 

2.2 Reagents/supplies

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

2.2.1 Carbonate coating buffer (pH 9.6) 

2.2.2 Phosphate-buffered saline (PBS) (pH 7.2) 

2.2.3 Phosphate-buffered saline with 0.05% Tween 20 (PBST) 

2.2.4 Blocking diluent (2% nonfat dry milk in PBS) 

2.2.5 Reagent diluent (2% nonfat dry milk in PBST) 

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Supplemental Assay Method for In vitro Potency Testing of Erysipelothrix rhusiopathiae Bacterins

2.2.6 Phosphate buffer for antigen elution (optional) 

2.2.7 0.5% sodium deoxycholate (desoxycholate) buffer for antigen elution 

(optional) 

2.2.8 Sodium citrate, reagent grade, for antigen elution (optional) 

2.2.9 3,3’,5,5’-tetramethylbenzine (TMB) substrate (Kirkegaard-Perry 

Laboratories, Inc., 2 Cessna Court, Gaithersburg, MD 20879, #50-76-00) 

2.2.10 Stop solution (2.5 M H2SO4) 

2.2.11 96-well flat-bottom microtitration plates (Immulon 2; Dynex Laboratories, 

Inc.)

2.2.12 96-well U-bottom, non-binding microtitration plates (transfer plate)

2.2.13 Plate sealers or Parafilm® 

2.2.14 Monoclonal antibody (MAb) against the 65-kD protein of 

E. rhusiopathiae, MAb ERHU1-B60-91. This reagent is available from the Center 

for Veterinary Biologics (CVB). Refer to the current reagent data sheet for 

additional information. 

2.2.15 Rabbit origin polyclonal antiserum against 65-kD protein of 

E. rhusiopathiae (monospecific). This reagent is available from the CVB. Refer to 

the current reagent data sheet for additional information. 

2.2.16 Horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) (Jackson 

ImmunoResearch Laboratories, Inc.) 

2.2.17 Qualified unexpired reference bacterin containing E. rhusiopathiae

CRITICAL CONTROL POINT: The reference and test serials should be adjuvanted 

with the same adjuvant. Ideally, the reference and test serials should be produced by the 

same Outline of Production. 

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Supplemental Assay Method for In vitro Potency Testing of Erysipelothrix rhusiopathiae Bacterins

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel should have working knowledge of the use of general laboratory 

chemicals, equipment, and glassware; performance of ELISA assays; and knowledge and 

experience in the operation of an automated microplate reader and data recording and 

evaluation software. 

3.2 Preparation of equipment/instrumentation

Operate and maintain all equipment according to manufacturers’ recommendations and 

applicable in-house standard operating procedures. 

3.3 Preparation of reagents

3.3.1 Carbonate coating buffer (0.05 M, pH 9.6) 

Na2CO3 0.159 g 

NaHCO3 0.293 g 

Deionized water q.s. to 100 mL 

Adjust pH to 9.6 ± 0.1. Store at 2°- 7°C for no longer than 1 week. 

3.3.2 Phosphate buffered saline (PBS) (pH 7.2) 

NaCl 8.00 g 

KCl 0.20 g 

Na2HPO4 1.15 g 

KH2PO4 0.20 g 

Deionized water q.s. 1 L 

Adjust pH to 7.2 ± 0.1 with 0.1 M NaOH or 0.1 M HCl.. Store at 20°- 25°C for no 

longer than 6 months. If the longer-term storage is desired, autoclave to sterilize 

before storage.

Caution: Concentrated solutions of acids and bases are used to prepare some of the 

following reagents. Both are hazardous and should be handled properly. Consult 

Material Safety Data Sheets (MSDS) (current version) for proper safety procedures.

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Supplemental Assay Method for In vitro Potency Testing of Erysipelothrix rhusiopathiae Bacterins

3.3.3 PBS-Tween 20 (PBST) 

 

PBS (see Section 3.3.2) 1 L 

Tween 20 0.50 mL 

Store at 20°- 25°C for no longer than 6 months. 

3.3.4 Blocking diluent (2% nonfat dry milk in PBS) 

Nonfat dry milk 4.0 g 

PBS (see Section 3.3.2) 200 mL 

Make fresh daily.

3.3.5 Reagent diluent (2% nonfat dry milk in PBST)

Nonfat dry milk 4.0 g 

PBST (see Section 3.3.3) 200 mL 

Make fresh daily. 

3.3.6 Phosphate buffer for antigen elution 

KH2PO4 8.2 g 

Deionized water q.s. to 100 mL 

Adjust pH to 6.5 ± 0.1. Store at 20°- 25°C for up to 30 days. 

3.3.7 0.5% sodium deoxycholate (desoxycholate) for antigen elution 

PBS (see Section 3.3.2) 100 mL 

Sodium deoxycholate (Sigma #D6750 or equivalent) 0.50 g 

Store at 2°- 7oC for no longer than 1 month. Warm to 20°- 25°C prior to use. 

Warning: This solution gels at 2°- 7°C.

3.4 Preparation of the sample 

Some bacterins do not require antigen-elution treatment prior to being tested in this assay. 

Representative serials should be tested with and without antigen-elution treatment to 

determine if the treatment specifically enhances the capture of 65-kD protein. If no 

enhancement of antigen capture can be demonstrated, the bacterins should be tested 

without antigen-elution treatment. The reference bacterin and the test serials for each 

product must be treated by the same procedure. The following section discusses some of 

the more common antigen elution treatments. However, alternative elution procedures 

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Supplemental Assay Method for In vitro Potency Testing of Erysipelothrix rhusiopathiae Bacterins

may be more appropriate for some bacterins. Antigen-elution treatments should be 

optimized for each manufacturer’s bacterin(s). Treat bacterins prior to making serial 

twofold dilutions in reagent diluent. 

3.4.1 Aluminum-adjuvanted bacterins

1. Sodium citrate elution

Add 1.0 g sodium citrate to 10.0 mL bacterin (10% w/v). Place on an 

orbital shaker (100-130 rpm) 16 to 24 hours at 35°- 38°C. Treated bacterin 

is considered undiluted. 

2. Phosphate buffer elution 

Add 1.0 mL of phosphate elution buffer to 1.0 mL of bacterin. Place on an 

orbital shaker (100-130 rpm) 16 to 24 hours at 35°- 38°C. Treated bacterin 

is considered diluted 1:2. 

3.4.2 Oil-adjuvanted bacterins 

Mix 1.0 mL 0.5% sodium deoxycholate with 1.0 mL bacterin. Place on an orbital 

shaker (100-130 rpm) 16 to 24 hours at 35°- 38°C. Treated bacterin is considered 

diluted 1:2. 

4. Performance of the Capture ELISA

4.1 Dilute MAb ERHU1-B60-91 to the current use dilution (refer to current reagent 

data sheet) in cold carbonate coating buffer. Place 100 μL diluted MAb into each well of 

a 96-well flat-bottom microtitration plate. Seal plates with adhesive plate sealers or 

Parafilm®. Incubate overnight at 2°- 7°C for at least 16 hours. Coated plates may be held 

at 2°- 7°C up to 5 days. 

4.2 Wash the plate 3 times with PBST. An automatic plate washer (200-250 μL/well, 

10- to 40-second soak cycle, 3 wash cycles) may be used, or the plates may be washed by 

hand. Tap the plates upside down on absorbent material to remove residual fluid. 

4.3 Place 100 μL blocking buffer in each well of the plate. Incubate at 35°- 38°C for 

60 to 75 minutes. 

4.4 Wash plate as described in Section 4.2. 

4.5 Make twofold dilutions of reference and test bacterins in a transfer plate. Place 

125 μL of reagent diluent into each well of a 96-well U-bottom microtitration plate. Add 

125 μL of bacterin to the first well of each of a minimum of 2 columns or rows per 

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Supplemental Assay Method for In vitro Potency Testing of Erysipelothrix rhusiopathiae Bacterins

bacterin. Use a multichannel pipetting device to make serial twofold dilutions of each 

bacterin across the plate. At least 2 wells must be reserved without bacterin for use as a 

blank. 

The use of at least 7 serial twofold dilutions is recommended. Ideally, the selected 

bacterin dilutions should delineate the sigmoid curve from antigen saturation to antigen 

extinction for each bacterin. The dilutions used for the reference and bacterin may differ. 

4.6 Transfer 100 μL diluted bacterin from each well of the transfer plate to the 

corresponding well on the test plate. Seal plate and incubate for 60 to 75 minutes at 

35°- 38°C. 

4.7 Wash plate as described in Section 4.2. 

4.8 Dilute polyclonal rabbit antiserum to current use dilution (refer to the current 

reagent data sheet) in reagent diluent. Place 100 μL diluted antibody in each well of the 

test plate. Seal plate, and incubate for 60 to 75 minutes at 35°- 38°C.

4.9 Wash plate as described in Section 4.2. 

4.10 Dilute anti-rabbit horseradish peroxidase conjugate in reagent diluent to 

appropriate use dilution as determined by checkerboard titration for that lot. Place 100 μL 

diluted conjugate in each well of the test plate. Seal plate and incubate for 60 to 75 

minutes at 35°- 38°C.

4.11 Measure out the necessary volume (10 mL/plate) of TMB substrate. Store in the 

dark during the conjugate incubation step to allow substrate to come to 20°- 25°C. 

4.12 Wash plate as described in Section 4.2. 

4.13 Place 100 μL TMB substrate solution into each well of the test plate. Incubate at 

20°- 25°C for 7 to 15 minutes, or until adequate color change has occurred. 

4.14 Add 100 μL stop solution (2.5M H2SO4) to each well of the plate.

4.15 Read the ELISA plate(s) at 450/650 nm. Calculate the mean absorbance for the 

blank wells. Subtract the mean absorbance of the blank wells from each bacterin well 

absorbance value prior to data analysis.

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Supplemental Assay Method for In vitro Potency Testing of Erysipelothrix rhusiopathiae Bacterins

5. Interpretation of the Test Results

5.1 Relative potency calculation method 

Refer to the current version of CVBSOP0102 for estimating the relative potency.

5.2 Requirements for a valid assay 

5.2.1 An assay must meet the validity requirements of CVBSOP0102 to be 

considered valid. 

5.2.2 Lines determined by first-order linear regression must have a correlation 

coefficient (r) of ≥ 0.95. 

5.2.3 The reference dose-response line and the test serial dose-response line 

must show parallelism.

5.2.4 Assays that are not valid may be repeated up to a maximum of 3 times. If 

a valid assay cannot be achieved with 4 independent assays, the serial shall be 

deemed unsatisfactory. 

5.3 Requirements for a satisfactory serial

To be considered satisfactory, a test serial must have an RP value of ≥ 1.0 in a valid 

assay. Serials with RP values < 1.0 on a valid assay may be retested by conducting 2 

independent replicate tests in a manner identical to the initial test. If the reported RP 

values of both of the retests are ≥ 1.0, the serial is satisfactory. If the test serial is not 

retested, the serial is unsatisfactory.

6. Report of Test Results 

Results of the test(s) are reported as described by standard operating procedures.

7. Summary of Revisions 

Version .05

• Updated coversheet and contact information. 

Version .04

• Section Leader and Director information updated. 

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Supplemental Assay Method for In vitro Potency Testing of Erysipelothrix rhusiopathiae Bacterins

• Relative Potency reference document changed from SAM 318 to CVBSOP0102. 

Version .03

• The Contact information has been updated. 

Version .02 

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

Biologics and to provide additional detail. No significant changes were made that impact the 

outcome of the test. 

• 2.2 The use of 2,2 ́-azino-di-3-ethylbenzithiazoline sulfonate (ABTS) substrate has 

been discontinued and this has been removed from the document. 

• Desoxycholate has been changed to deoxycholate (the two chemicals are equivalent).

• Reference to the current reagent data sheet has been added throughout the document 

as appropriate. 

• The title of this document has been revised to clarify that this SAM describes an invitro assay, not the codified mouse assay (described in SAM 611). 

• The contact person has been changed to Janet Wilson.

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