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Supplemental Assay Method for Potency Testing Enterotoxigenic (F41 Pilus) Escherichia coli Bacterins

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 623

Date: May 26, 2023

Number: SAM 623.07

Supersedes: SAM 623.06, May 9, 2017

Standard Requirement: Approved, pending 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 Potency Testing Enterotoxigenic (F41 Pilus) Escherichia coli Bacterins

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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

5.1 Relative potency calculation method

5.2 Requirements for a valid assay

5.3 Requirements for a satisfactory test bacterin

6. Reporting of Test Results

7. Summary of Revisions
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Supplemental Assay Method for Potency Testing Enterotoxigenic (F41 Pilus) Escherichia coli Bacterins

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1. Introduction

This Supplemental Assay Method (SAM) for potency testing inactivated Escherichia coli

bacterins employs a capture enzyme-linked immunosorbent assay (ELISA) for F41 pilus antigen. 

Relative potency is determined by comparing the F41 antigen content of the test bacterin to the 

F41 antigen content present in an unexpired, 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 (490 nm and 650 nm)

2.1.2 Automatic microplate washer (optional)

2.1.3 Micropipettors, to cover the range of 5-μL to 1000-μL

2.1.4 8- or 12-channel micropipettor, to cover the range of 50-μL to 200-μL

2.1.5 Orbital shaker

2.1.6 Balance, to measure 150 mg to 15 g

2.1.7 Relative Potency Calculation Software 

2.2 Reagents/supplies

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

2.2.1 96-well flat-bottom, high-binding microtitration plates (Immulon 2; 

Dynatech Laboratories, Inc.)

2.2.2 96-well non-binding microtitration plates suitable for making serial 

dilutions (transfer plates)

2.2.3 Plate sealers
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2.2.4 Carbonate coating buffer 

2.2.5 Phosphate-buffered saline (PBS), pH 7.2

2.2.6 Phosphate-buffered saline with 0.05% Tween 20 (PBS-Tween)

2.2.7 PBS-Tween with 2.0% bovine albumin fraction V (monoclonal antibody 

(MAb) diluent)

2.2.8 PBS-Tween with 1.0% normal rabbit serum (conjugate diluent)

2.2.9 Phosphate elution buffer (optional)

2.2.10 Sodium citrate, dihydrate for antigen elution (optional)

2.2.11 Sodium desoxycholate elution buffer (optional)

2.2.12 Citrate buffer (substrate diluent)

2.2.13 o-Phenylenediamine dihydrochloride (OPD)

2.2.14 Hydrogen peroxide, 30%, stabilized

2.2.15 2.5M H2SO4 stop solution

2.2.16 F41-specific antigen-capture polyclonal antibody (F41 PAb), rabbit origin

(available from the Center for Veterinary Biologics (CVB)). Refer to the current 

reagent data sheet for details.

2.2.17 F41-specific antigen-indicator MAb, SDSU 56/85 (F41 MAb) (available 

from the CVB). Refer to the current reagent data sheet for details.

2.2.18 Horseradish peroxidase-conjugated goat anti-mouse IgG (H+L) obtained

from a commercial source (Jackson ImmunoResearch Laboratories, Inc., 

115-035-062)

2.2.19 Test bacterin(s) containing F41 antigen

2.2.20 Reference bacterin containing F41 antigen (must be approved by the 

Animal and Plant Health Inspection Service and within dating)
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Supplemental Assay Method for Potency Testing Enterotoxigenic (F41 Pilus) Escherichia coli Bacterins

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3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel need a working knowledge of the use of general laboratory 

chemicals, equipment, and glassware; automated microplate washer and reader; and data 

recording and evaluation software. They need specific training in the performance of this 

assay.

3.2 Preparation of equipment/instrumentation

Operate and maintain all equipment according to manufacturers’ recommendations and 

applicable standard operating procedures. 

3.3 Preparation of reagents/control procedures

3.3.1 Carbonate coating buffer – National Centers for Animal Health (NCAH)

Media #20034 

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) – NCAH Media #10559 

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. Store at 20°- 25°C. If long-term storage (up to 1 year) is 

desired, autoclave 20-30 minutes at ≥ 121°C to sterilize following manufacturer’s 

recommendations. 

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

the following reagents. Both are hazardous and must be handled properly. Consult 

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

procedures.
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3.3.3 Phosphate-buffered saline with 0.05% Tween 20 (PBS-Tween) – NCAH

Media #30179

PBS (see Section 3.3.2) 1 L

Tween 20 0.5 mL

Store at 20°- 25°C for no longer than 1 year.

3.3.4 PBS-Tween with 2.0% bovine albumin fraction V (MAb diluent)

PBS-Tween (see Section 3.3.3) 25 mL

Bovine albumin fraction V (Scientific Protein 

 Laboratories, Viobin Corp., Waunakee, WI,

 40-6197-2-1160 or equivalent) 0.5 g

Prepare immediately prior to use. Swirl gently to dissolve the powder.

3.3.5 PBS-Tween 20 with 1.0% normal rabbit serum (conjugate diluent)

PBS-Tween 20 (see Section 3.3.3) 24.75 mL

Normal rabbit serum

 (negative for E. coli antibodies) 0.25 mL

Prepare immediately prior to use. Swirl gently to mix.

3.3.6 Phosphate elution buffer 

KH2PO4 (reagent grade) 8.2 g

Deionized water 94 mL

Adjust pH to 9.3 ± 0.1, or other appropriate pH as optimized for use with a 

specific bacterin. Store at 20°- 25°C for no longer than 1 month.

3.3.7 Sodium deoxycholate (desoxycholate) elution buffer

Sodium deoxycholate (reagent grade) 0.50 g

PBS (see Section 3.3.2) 100 mL

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

buffer gels at 2°- 7°C.)
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3.3.8 Citrate buffer (pH 5.0) – NCAH Media #20033 

Citric acid monohydrate (reagent grade) 5.26 g 

Na2HPO4•7H2O 6.74 g 

Deionized water q.s. to 1 L 

Adjust pH to 5.0 ± 0.1 and filter sterilize. Store at 2°- 7°C for no longer than 2 

months. Use to prepare substrate solution (Section 3.3.9).

 

3.3.9 Substrate solution (quantities for 1 plate) 

Citrate buffer 12 mL 

o-Phenylenediamine dihydrochloride (OPD) 

 (Sigma P8787 or equivalent) 4 mg 

30% H2O2 (stabilized) 5 μL 

Prepare within 15 minutes of use. 

3.3.10 Stop solution (2.5 M H2SO4) – NCAH Media #30171 

Concentrated (98%) H2SO4 13.6 mL 

Deionized water 86.4 mL 

Add acid to water. Solution may be stored no longer than 1 year at 20°- 25°C. 

3.3.11 Bacterins containing F41 antigen 

1. Reference bacterin

2. Test bacterin(s)

CRITICAL CONTROL POINT: Ideally, the reference and test bacterins should 

be produced by the same Outline of Production. If reference formulation differs 

from that of the test bacterin, the assay must be validated to show that this does 

not adversely affect assay performance or accuracy of results.

Caution: o-Phenylenediamine dihydrochloride is a carcinogen. See 

appropriate MSDS for precautions when handling this product.
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3.4 Preparation of the sample

Antigen-elution treatments: Many bacterins do not require antigen-elution treatment prior 

to being serially diluted in twofold increments with PBS-Tween 20. Test representative 

batches of each adjuvanted product with and without each antigen-elution treatment to 

determine if the treatment specifically enhances F41 antigen capture. If no enhancement 

of F41 antigen capture can be demonstrated, test the bacterins without antigen-elution 

treatment. Treat the reference bacterin and the test bacterins for each product by the same 

procedure. Alternative elution procedures, other than those described here, may be more 

appropriate for some bacterins.

3.4.1 Aluminum-adjuvanted bacterins

Bacterins adjuvanted with aluminum hydroxide may be treated with either sodium 

citrate or phosphate elution buffer prior to making serial twofold dilutions in 

PBS-Tween 20.

• Sodium citrate elution

Add 1.0 g sodium citrate, dihydrate to 10.0 mL of bacterin (10% w/v). 

Place on an orbital shaker (100-120 rpm) overnight at 35°- 37°C. 

Consider treated bacterin to be undiluted.

• Phosphate buffer elution

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

on an orbital shaker (100-120 rpm) overnight at 35°- 37°C. Consider 

treated bacterin to be diluted 1:2.

3.4.2 Oil-adjuvanted bacterins

Mix 1.0 mL of the sodium deoxycholate elution buffer with 1.0 mL of bacterin. 

Place on an orbital shaker (100-120 rpm) overnight at 35°- 37°C. Consider treated 

bacterin to be diluted 1:2.

4. Performance of the Test

4.1 Dilute the F41 PAb in cold carbonate coating buffer (refer to the current reagent 

data sheet for the current use dilution). Place 100 μL in each well of a 96-well flatbottom, high-binding microtitration plate. Seal coated plates with plate sealers. Incubate 

the coated plates overnight at 2°- 7°C. Store sealed plates at 2°- 7°C for no longer than 5 

days.
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4.2 Make twofold dilutions of reference and test bacterins, using PBS-Tween 20 as a 

diluent. Add 125 μL PBS-Tween 20 to each well of a clean microtitration plate (transfer 

plate). Place 125 μL of bacterin in the first well of each row. Test each bacterin in at least 

2 replicate rows. Test the reference bacterin and the test bacterin on the same plate. 

Use a multichannel micropipettor to make serial twofold dilutions of each bacterin across 

the plate (125 μL transfer volume). Reserve at least 2 unused wells on each plate to serve 

as blanks. The use of at least 7 serial twofold dilutions per bacterin 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 

the test bacterin may differ.

4.3 Wash the coated ELISA plates 3 times with PBS-Tween 20. An automatic plate 

washer (200-300 μL/well, 10- to 40-second soak cycle) may be used. Alternatively, 

plates may be hand washed. Tap the plates upside down on absorbent material to remove 

residual fluid.

4.4 Use a multichannel micropipettor to transfer the bacterin dilutions from the 

transfer plates to the coated ELISA plates (100 μL/well). Seal the ELISA plates and 

incubate them on an orbital shaker (100-120 rpm) for 30 minutes (± 5 minutes) at 

20°- 25°C.

4.5 Wash the ELISA plates 3 times with PBS-Tween 20 as in Section 4.3.

4.6 Dilute the F41 MAb in MAb diluent to the appropriate use dilution (refer to the

current reagent data sheet for dilution) and add 100 μL to each well. Seal the ELISA 

plates and incubate on an orbital shaker (100-120 rpm) for 30 minutes (± 5 minutes) at 

20°- 25°C.

4.7 Wash the ELISA plates 3 times with PBS-Tween 20 as in Section 4.3.

4.8 Dilute the peroxidase-conjugated goat anti-mouse IgG in conjugate diluent to the 

appropriate use dilution and add 100 μL to each well. Seal the ELISA plates and incubate 

on an orbital shaker (100-120 rpm) for 30 minutes (± 5 minutes) at 20°- 25°C.

4.9 Wash the ELISA plates 3 times with PBS-Tween 20 as in Section 4.3.

4.10 Add 100 μL substrate solution to each well. Incubate the ELISA plates on an 

orbital shaker (100-120 rpm) for 10 minutes (± 5 minutes) or until sufficient color 

develops at 20°- 25°C.

4.11 Stop the substrate color development by adding 100 μL stop solution to each well.

Note: The OPD substrate buffer undergoes a color shift from yellow to orange when 

stop solution is added.
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4.12 Read the ELISA plates using an ELISA reader with dual wavelengths (490 nm 

test, 650 nm reference). Calculate the mean absorbance for the blank wells. Subtract the 

mean absorbance of the blank wells from each bacterin test well absorbance value prior 

to data analysis.

5. Interpretation of the Test Results

5.1 Relative potency calculation method

5.1.1 Calculate the relative potency of the test bacterin as compared to that of 

the reference bacterin as described by standard operating procedure, 

CVBSOP0102, Using Software to Estimate Relative Potency.

5.1.2 Do not use bacterin dilutions with mean absorbance values less than 

(<) 0.05 (after subtraction of the mean absorbance of the blank) in the relative 

potency calculations. 

5.1.3 Do not use regression lines with slopes less than (<) 0.10 in minimum 

absolute value for relative potency calculations.

5.2 Requirements for a valid assay

5.2.1 Lines are determined by first-order linear regression of at least 3 

contiguous points and must have a correlation coefficient (r) of greater than or 

equal to (≥) 0.95.

5.2.2 The reference regression line and the test bacterin regression line must 

show parallelism (slope ratio 0.80 to 1.25).

5.3 Requirements for a satisfactory test bacterin

To be considered satisfactory, a test bacterin must have an RP value of ≥ 1.0. Test 

bacterins 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 both retests are valid 

and the reported RP values of both of the retests are ≥ 1.0, the test bacterin is satisfactory.

6. Reporting of Test Results

Report results of the test(s) as described by standard operating procedures.
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7. Summary of Revisions 

Version .07 

• Updated coversheet and contact information.

Version .06 

• Updated minimum slope expectations for consistency with the PEL Reviewer’s 

Manual Work Instruction 4.6.2.

Version .05

• Bacteriology Section Leader has been updated. 

• Clarified media expiration dates.

• Removed references to Relative Potency Calculation Software (RelPot) and 

associated SAM 318. 

Version .04

• The Contact information has been updated. 

• All references to National Veterinary Services Laboratories media have been changed 

to National Centers for Animal Health media.

Version .03

• Clarification that sodium citrate, dihydrate should be used (rather than sodium citrate 

monobasic, anhydrous) has been added throughout the document. 

Version .02

This document was revised to clarify 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: 

• 2.2.1 The assay plate has been further described as high-binding. 

• 2.2.2 The transfer plate has been further described as non-binding. 

• 2.2.15 and 3.3.10 The stop solution has been further defined and storage information 

has been updated. 

• 3.3.5 The formula has been updated, including the specification that the serum not 

contain E. coli antibodies. 

• 3.3.7 The alternate chemical name has been included.

• 3.3.8 Filter sterilization of the solution has been added. 

• 4.10 Additional details for stopping the reaction have been added. 

• References to the current reagent data sheet have been added throughout the 

document. 

• References to internal CVB documents have been replaced with summary 

information. 

• The contact person has been changed to Janet M. Wilson.