SAM 627.08

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 627

Supplemental Assay Method for In vitro Potency Testing of Leptospira 

interrogans serogroup icterohaemorrhagiae Bacterins

Date: May 28, 2023 Approved, pending Standard Requirement

Number: SAM 627.08

Supersedes: SAM 627.07, April 28, 2017

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.
SAM 627.08

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Supplemental Assay Method for In vitro Potency Testing of Leptospira 

interrogans serogroup icterohaemorrhagiae 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 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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1. Introduction

This Supplemental Assay Method (SAM) uses a sandwich enzyme-linked immunosorbent assay 

(ELISA) to measure the relative potency of bacterins containing Leptospira interrogans

serogroup icterohaemorrhagiae (except strain bogvere) compared to a suitably qualified, 

nonexpired 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 5.0-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 (405 nm and 490 nm)

2.1.6 Balance, validated from 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 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 buffer for elution (optional)

2.2.6 Sodium citrate for elution (optional)
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2.2.7 Sodium deoxycholate (desoxycholate) elution buffer (optional)

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

2.2.9 Antibody diluent with normal rabbit serum (negative for Leptospira spp.)

2.2.10 ABTS (2,2’-azino-di-3-ethylbenziazoline sulfonate) substrate, 1- or 2-

component (Kirkegaard and Perry Laboratories, Inc.)

2.2.11 L. icterohaemorrhagiae monoclonal antibody (LI MAb) is available from 

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

for additional information.

2.2.12 Polyclonal L. icterohaemorrhagiae antiserum (LI PAb) of rabbit origin. 

L. icterohaemorrhagiae PAb is available from the CVB. Refer to the current 

reagent data sheet for additional information.

2.2.13 Goat anti-mouse IgG (H+L) horseradish peroxidase-labeled antibody, 

Human Serum Adsorbed (Kirkegaard and Perry Laboratories, Inc.)

2.2.14 Test bacterin(s) containing L. icterohaemorrhagiae

2.2.15 Reference bacterin (nonexpired) containing L. icterohaemorrhagiae (must 

be approved by the Animal and Plant Health Inspection Service.

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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3.3 Preparation of reagents

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

Media #50178

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 up to 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. Autoclave at ≥ 121oC for 20 minutes. Store at 20°- 25°C 

for no longer than 6 months.

3.3.3 Polyvinyl alcohol, 1%, in PBS

PBS (see Section 3.3.2) 70 mL

Polyvinyl alcohol, 87-89% hydrolyzed, 

MW 13,000-23,000 (Sigma-Aldrich, 

 Cat. No. 363170, or equivalent) 0.7 g

Stir to dissolve using low heat if needed. Sterile filter and store at 20°- 25°C for 

no longer than 3 months.

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

NCAH Media #30179 

PBS (see Section 3.3.2) 1000 mL

Tween 20 0.50 mL

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

3.3.5 Antibody diluent with normal rabbit serum

Polyvinyl alcohol, 1%, in PBS (see Section 3.3.3) 43.6 mL

Normal rabbit serum 400 μL

Mix immediately prior to use. Store at 2°- 7°C for up to 1 week.
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3.3.6 Phosphate buffer for antigen elution 

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. 

3.3.7 Sodium deoxycholate (desoxycholate) elution buffer 

Sodium deoxycholate (Sigma Chemical, 

 D6750, or equivalent) 0.50 g 

PBS (see Section 3.3.2) 100 mL

Store at 2°- 7°C for up to 30 days. Warm to 20°- 25°C prior to use (buffer gels at 

2°- 7°C).

3.3.8 LI MAb 

Obtain LI MAb from the CVB. Store at 2°- 7°C for several weeks or store longterm at -70°C or colder. Refer to the current reagent data sheet for additional 

details.

Note: LI MAb does not recognize antigens in L. icterohaemorrhagiae serovar 

bogvere (type strain LT60-69). Bacterins containing serovar bogvere must be 

assayed by an alternative procedure.

Obtain the rabbit antiserum from the CVB. Store at 2°- 7°C for several weeks or 

store long term at -70°C or colder. Refer to the current reagent data sheet for 

additional details.

3.3.9 Bacterins containing L. icterohaemorrhagiae 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

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 
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product with and without each antigen-elution treatment to determine if the treatment 

specifically enhances antigen capture. If no enhancement of 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. Alternative 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. 

1. Sodium citrate elution

Mix 1 g sodium citrate with 10 mL of bacterin (10% w/v). Place on an 

orbital shaker (100-120 rpm) overnight at 36°- 38°C. Consider treated 

bacterin to be undiluted.

2. Phosphate buffer elution

Mix 1 mL of phosphate elution buffer with 1 mL of bacterin. Place on an 

orbital shaker (100-120 rpm) overnight at 36°- 38°C. Consider treated 

bacterin to be diluted 1:2.

3.4.2 Oil-adjuvanted bacterins

Mix 1 mL of sodium deoxycholate (desoxycholate) elution buffer with 1 mL of 

bacterin. Place on an orbital shaker (100-120 rpm) overnight at 20°- 25°C. 

Consider treated bacterin to be diluted 1:2.

4. Performance of the Test

4.1 Dilute L. icterohaemorrhagiae rabbit antiserum to the current use dilution (refer 

to current reagent data sheet) in cold carbonate coating buffer. Dispense 100 μL into each 

well of a 96-well microtitration plate (test plate). Seal plate and incubate at 2°- 7°C for 16

to 20 hours. Coated plates may be stored at 2°- 7°C 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 pipetting device 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. 
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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 bacterin and the 

test bacterin may differ.

4.3 Wash the test plate(s) once with PBS-Tween 20. An automatic plate washer 

(200 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.

4.4 Use a multichannel pipetting device to transfer the bacterin dilutions from the 

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

incubate the plates for 60 to 90 minutes at 36°- 38°C.

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

4.6 Dilute LI MAb to the current use dilution (refer to current reagent data sheet) in 

antibody diluent, and add 100 μL to each well of the test plate. Incubate the plates for 55

to 65 minutes at 36°- 38°C.

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

4.8 Dilute the goat anti-mouse IgG horseradish peroxidase conjugate 1:2000 in 

antibody diluent. Alternatively, the appropriate use dilution may be specified by the

manufacturer or determined by checkerboard titration for that lot. Add 100 μL to each 

well of the test plate. Incubate for 30 to 60 minutes at 36°- 38°C.

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

4.10 Add ABTS substrate (100 μL) to all wells, and incubate plates for 15 to 30 

minutes at 36°- 38°C. 

4.11 Read plates at 405/490 nm. 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.
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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. 

5.2 Requirements for a valid assay

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

points 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).

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

a valid assay cannot be achieved with 3 independent assays, the test bacterin is 

unsatisfactory. 

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. Report of Test Results

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

7. Summary of Revisions 

Version .08 

• Updated title page and formatting.

• Removed references to 294-004 clone for LI MAb source and instead direct to 

reagent data sheet for most current information. 

• Updated carbonate coating buffer NCAH Media number to 50178. 

• Updated Polyvinyl alcohol description. 
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Version .07 

• Updated minimum slope expectations for consistency with PEL Reviewer Manual 

Work Instruction 4.6.2.

Version .06 

• The Bacteriology Section Leader and PEL Director were updated.

• References to Relative Potency Calculation Software (RelPot) and associated SAM 

318 were removed. 

Version .05

• The contact person was changed to Angela M. Walker. 

• Minor changes were made to clarify practices currently in use at CVB.

Version .04

• Additional conjugate information has been added to reflect current procedures. 

Version .03

• Minor changes have been made to update the document to current formatting 

practices.

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: 

• 2.2.7 Desoxycholate changed to deoxycholate to reflect Sigma catalog listing (the 

two chemicals are equivalent).

• 4.2 Test well replicates changed from 3 to 2. 

• References to the current reagent data sheet were added throughout the document as 

appropriate. 

• The contact has been changed to Heather C. Hutchings.