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Supplemental Assay Method for Potency Testing Enterotoxigenic (K88 Pilus) Escherichia coli Bacterins

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 621

Date: May 26, 2023

Number: SAM 621.07

Supersedes: SAM 621.06, May 10, 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 (K88 Pilus) Escherichia coli 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/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. Report of Test Results

7. Summary of Revisions
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Supplemental Assay Method for Potency Testing Enterotoxigenic (K88 Pilus) Escherichia coli Bacterins

1. Introduction

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

bacterins employs a capture enzyme-linked immunosorbent assay (ELISA) for K88 pilus 

antigen. Relative potency is determined by comparing the K88 antigen content of the test 

bacterin to the K88 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 Micropipettors, to cover the range of 5-μL to 1000-μL

2.1.2 8- or 12-channel micropipettor, to cover the range of 50-μL to 200-μL

2.1.3 Orbital shaker

2.1.4 Automatic microplate washer (optional)

2.1.5 Microplate reader with dual wavelengths (490 nm and 650 nm)

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 plate)

2.2.3 Plate sealers

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)
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Supplemental Assay Method for Potency Testing Enterotoxigenic (K88 Pilus) Escherichia coli Bacterins

2.2.7 PBS-Tween with 1.0% ovalbumin (conjugate diluent)

2.2.8 Phosphate elution buffer (optional)

2.2.9 Sodium citrate, dihydrate for antigen elution (optional)

2.2.10 Sodium desoxycholate elution buffer (optional)

2.2.11 Citrate buffer (substrate diluent)

2.2.12 o-Phenylenediamine dihydrochloride (OPD)

2.2.13 Hydrogen peroxide (H2O2), 30%, stabilized

2.2.14 2.5 M H2SO4 Stop solution

2.2.15 K88-specific antigen-capture monoclonal antibody (available from the 

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

for details.

2.2.16 Horseradish peroxidase-conjugated K88-specific monoclonal antibody

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

2.2.17 Test bacterin(s) containing K88 antigen

2.2.18 Reference bacterin containing K88 antigen (approved by the Animal and 

Plant Inspection Service and within dating).

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 microplate 

reader; and data analysis 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 in-house standard operating procedures.
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Supplemental Assay Method for Potency Testing Enterotoxigenic (K88 Pilus) Escherichia coli Bacterins

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. to 1 L

Adjust pH to 7.2 ± 0.1. Store at 20°- 25°C for no longer than 6 months. If long 

term storage (up to 1 year) is desired, autoclave for 20-30 minutes at ≥ 121°C to 

sterilize following manufacturer’s recommendations.

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 no longer than 1 year.

3.3.4 PBS-Tween with 1.0% ovalbumin (conjugate diluent)

PBS-Tween (see Section 3.3.3) 20 mL

Ovalbumin (Calbiochem 32467 or equivalent) 0.2 g

Add ovalbumin to the PBS-Tween 20 within 10 minutes of use. Swirl gently to 

dissolve the crystals. 
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Supplemental Assay Method for Potency Testing Enterotoxigenic (K88 Pilus) Escherichia coli Bacterins

3.3.5 Phosphate elution buffer 

KH2PO4 (Mallinckrodt, Inc. 7100 or equivalent) 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. 

3.3.6 Sodium deoxycholate (desoxycholate) elution buffer 

Sodium desoxycholate 

 (Sigma Chemical D6750 or equivalent) 0.5 g 

PBS (see Section 3.3.2) 100 mL 

Store at 2°- 7°C for no longer than 1 month. Warm to room temperature prior to 

use; (the buffer gels at 2°- 7°C).

3.3.7 Citrate buffer (pH 5.0) – NCAH Media #20033 

Citric acid monohydrate (reagent grade) 

 (Fisher Scientific A104-500 or equivalent) 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 (see Section 3.3.8). 

3.3.8 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 

Caution: o-Phenylenediamine dihydrochloride is a carcinogen. See 

appropriate MSDS for precautions when handling this product.
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Supplemental Assay Method for Potency Testing Enterotoxigenic (K88 Pilus) Escherichia coli Bacterins

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

Concentrated (98%) H2SO4 13.6 mL

Deionized water 86.4 mL

Add acid to water. Store at 20°- 25°C for no longer than 1 year.

3.3.10 Monoclonal antibodies (MAb)

1. K88 antigen-capture monoclonal antibody (21BA1-1H1). Obtain MAb 

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

and storage.

2. Horseradish peroxidase-labeled K88 antigen-indicator MAb (21BA1-

1H1). Obtain conjugated MAb from the CVB. Refer to the current reagent 

data sheet for details on use and storage.

3.3.11 Bacterins containing K88 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.

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 the K88 antigen capture. If no 

enhancement of the K88 antigen capture can be demonstrated, test the bacterins without 

antigen-elution treatment. Treat the reference bacterin and the test bacterins by the same 

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

more appropriate for some bacterins.

3.4.1 Aluminum hydroxide adjuvanted bacterins

 

Bacterins adjuvanted with aluminum hydroxide may be treated with either 

sodium citrate or phosphate buffer prior to making serial twofold dilutions 

in PBS-Tween 20.
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Supplemental Assay Method for Potency Testing Enterotoxigenic (K88 Pilus) Escherichia coli Bacterins

• Sodium citrate elution

Mix 1.0 g sodium citrate, dihydrate with 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

Mix 1.0 mL of phosphate elution buffer with 1.0 mL 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 sodium desoxycholate 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 K88 antigen-capture MAb in cold carbonate coating buffer (refer to the 

current reagent data sheet for dilution) and place 100 μL in each well of 96-well flatbottom high-binding microtitration plates. Seal coated plates with plate sealers. Incubate 

coated plates overnight at 2°- 7°C. Coated plates stored at 2°- 7°C may be used for up to 

5 days.

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, or the plates may be 

washed by hand. 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 
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Supplemental Assay Method for Potency Testing Enterotoxigenic (K88 Pilus) Escherichia coli Bacterins

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 horseradish peroxidase-labeled K88 antigen-indicator MAb in 

conjugate diluent to the current 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.

Note: Ovalbumin will stick to plastic. Prepare the working dilution of the conjugate 

in a glass vial. Transfer the diluted conjugate into pipetting trays immediately prior 

to the addition of the conjugate to the ELISA plate.

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

4.8 Add 100 μL freshly prepared 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 is observed at 20°- 25°C.

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

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

solution is added.

4.10 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 procedures, 

CVBSOP0102, Using Software to Estimate Relative Potency.

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

0.050 (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.
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Supplemental Assay Method for Potency Testing Enterotoxigenic (K88 Pilus) Escherichia coli Bacterins

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

5.3.1 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. 

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

• The 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. 
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Supplemental Assay Method for Potency Testing Enterotoxigenic (K88 Pilus) Escherichia coli Bacterins

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. 

• 2.2.14 The Stop Solution used has been further described.

• 2.2.18 Further description of the reference bacterin as unexpired has been added. 

• 3.3.2 Additional information for long term storage has been added.

• 3.3.6 The alternate chemical name has been included. 

• 3.3.7 Filter sterilization of the solution has been added. 

• 3.3.9 The information on solution storage has been updated. 

• 3.4.1.2 The incubation temperature has been altered from 20°- 25°C to 35°- 37°C. 

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

• References to internal CVB documents have been replaced with summary 

information. 

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

document. 

• The contact person has been changed to Janet Wilson.