Salvaged Portions of Veterinary Services Memorandum No. 800.90 1

HOLD-OVER INFORMATION FROM VETERINARY SERVICES 

MEMORANDUM NO. 800.90

Industry has requested that some portions of obsoleted VSM 800.90 be preserved until 

the required information can be put into a rewrite of VSM 800.211. Therefore, the 

information below is included in the Reviewers’ Manual on an interim basis.

I. INTRODUCTION

These guidelines provide parameters and procedures for conducting an enzyme-linked 

immunosorbent assay (EIA or ELISA) for the quantitation of antigen in a veterinary 

biological product. The guidelines also establish uniformity for such in vitro testing.

In the ELISA or EIA system described, the concentration of a test serial is determined by 

comparing the absorbance values (optical density or OD) of that serial to the OD values 

of a reference preparation (Master or Working Reference). The relative potency of the 

test serial is determined from the relationship of the OD’s. These guidelines address the 

following: definitions, immunogen test specificity, in vitro test design, reference 

qualification, reference requalification, Qualifying Serials, reference storage, reference 

dating, testing adjuvanted product, and statistical methods.

II. DEFINITIONS

A. Applicable 9 CFR definitions include:

1. Master Reference, 9 CFR 101.5 (o). A Master Reference is a reference 

whose potency is correlated, directly or indirectly, to host animal 

immunogenicity. The Master Reference may be used as the working 

reference in in vitro tests for relative potency. The Master Reference may 

also be used to establish the relative potency of a serial of product used in 

requalification studies and to establish the relative potency of working 

references. The Master Reference as described in a filed Outline of 

Production may be:

a. A product reference that is a completed serial of vaccine or bacterin 

prepared in accordance with a filed Outline of Production. Product 

references may be:

i. Monovalent references composed of a single fraction or agent, 

or

ii. Polyvalent references composed of two or more fractions or 

agents

b. A purified preparation of a protective immunogen or antigen, or

c. A nonadjuvanted harvested culture of microorganisms.

2. Working Reference, 9 CFR 101.5 (p). A Working Reference is the reference 

preparation that is used in the in vitro test for the release of serials of product. 
Salvaged Portions of Veterinary Services Memorandum No. 800.90 2

Working References may be:

a. Master References, or

b. Serials of product that have been prepared and qualified, in a manner 

acceptable to the Animal and Plant Health Inspection Service 

(APHIS), for use as reference preparations.

3. Qualifying Serial, 9 CFR 101.5 (q). A Qualifying Serial is a serial of 

biological product used to test for immunogenicity when the Master or 

Working Reference is a purified antigen or nonadjuvanted harvest material. 

Qualifying Serials shall be produced in accordance with the filed Outline of 

Production, tested for immunogenicity in accordance with methods deemed 

appropriate by APHIS, and have a geometric mean relative potency, when 

compared to the Master Reference, of not greater than 1.0 as established by 

independent parallel line assays with five or more replicates or by other valid 

assay methods for determining the relative antigen content which 

demonstrate linearity, specificity, and reproducibility at least equivalent to 

the parallel line assay and are acceptable to APHIS. Qualifying Serials used 

to requalify or extend the dating period of a Master Reference shall be 

determined to be immunogenic in accordance with methods deemed 

appropriate by APHIS and, in addition, shall be within their permitted dating 

period and have been prepared in accordance with the production method 

described in the currently filed Outline of Production.

4. Immunogenicity, 9 CFR 101.5 (r). The ability of a biological product to 

elicit an immune response in animals as determined by test methods or 

procedures acceptable to APHIS.

III. DETERMINING THE TARGET OF THE TEST SYSTEM

A. Prior to the establishment of a testing method in the filed Outline of Production 

or Special Outline, the following should be supplied to and approved by APHIS:

1. Evidence that the in vitro relative potency test measures a protective 

immunogen as demonstrated by one of the following methods:

a. Host animal studies using a purified subunit (e.g. glycoprotein or 

bacterial outer membrane antigen extract) that elicits protection 

against the specific animal disease,

b. Passive protection by the monospecific antibody component(s) of the 

in vitro test system,

c. Data published in peer reviewed scientific journals generally 

recognized by the scientific community and acceptable to APHIS,

d. Demonstration of in vitro neutralization of viable organisms by the 

detecting reagent, or

e. Other methods acceptable to APHIS.
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2. Demonstration of the specificity of the reaction between a protective 

immunogen and the detection antibody by polyacrylamide gel 

electrophoresis/immunoblotting or other similarly specific techniques.

B. In general, the preferred ELISA or EIA potency test system uses a monoclonal 

antibody or mono-specific polyclonal antibody for a protective immunogen.

IV. REQUIREMENTS FOR A REFERENCE

A. The antigen content of a Master Reference should directly or indirectly correlate 

to protection of the host animal or support label claims.

1. A direct correlation is established when the Master Reference is used in the 

host animal protection study.

2. An indirect correlation is established when a Qualifying Serial is tested in 

host animals or when a Master Reference or Qualifying Serial is administered 

to animals other than host animals.

B. The reference used in the in vitro test for serial release is by definition a 

Working Reference by can, in addition, be the Master Reference.

C. It is recommended that the Master Reference and/or Working Reference be a 

product reference.

1. A product reference will have all the components in the same relative 

proportions as are found in the serial of product being tested.

2. A product reference can be either a monovalent reference or a polyvalent 

reference.

a. If a monovalent reference is used to evaluate a polyvalent product, it 

is unacceptable to compensate mathematically for in vitro interference 

occurring between fractions in the test serial.

b. If using a polyvalent reference to test product with fewer fractions 

than the reference, it is necessary to demonstrate that in vitro

interference between fractions is not occurring. If in vitro

interference occurs, then a monovalent reference or reference with 

similar antigen content is required.

D. Purified references are acceptable, provided that:

1. The test system is not influence by the other components present in the 

normal product, 

2. Linearity and parallelism between the purified Master or Working References 

and the product being tested are maintained,
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3. A Qualifying Serial is used to establish the purified Master Reference, and

4. There is no selective addition of components or compounds to a purified 

reference to compensate for nonlinearity or lack of parallelism.

E. Master References should be established to have a relative potency (RP) of one 

(1.0). This may be accomplished as follows:

1. For nonfrozen product references the RP of 1.0 is based on the concentration 

of antigen in the immunogenicity serial which demonstrated a protective 

response or supports label claims in a host animal immunogenicity trial.

2. For purified concentrated Master or Working References (frozen or nonfrozen), the dilution of the reference equivalent to an RP of 1.0 is established 

at the time of reference qualification using a Qualifying Serial which 

demonstrates a protective response or supports label claims in a host animal 

immunogenicity trial.

a. Dilution of the purified Master or purified Working Reference prior to 

use in the test system should not exceed 1:100 and should be done 

with an inert-ingredient diluent (phosphate buffered saline, 

Dulbecco’s phosphate buffered saline, physiological saline, water, 

etc.).

3. Frozen product Master Reference should be established to have an RP of 1.0 

and validated during qualification by the use of a qualifying product serial 

demonstrating a protective response or supporting label claims in a host 

animal immunogenicity trial.

a. Dilution of a product Master Reference with an inert ingredient 

diluent (phosphate buffered saline, Dulbecco’s phosphate buffered 

saline, physiological saline, water, etc.) to compensate for a 

difference in dose is allowed.

F. It is recommended that a new vial of reference be used for each test. Vials of 

frozen references should not be refrozen or used for more than one (1) day unless 

the additional time or freeze-thaw cycles are supported by data submitted and 

approved by APHIS and the criteria specified in the Outline of Production or 

Special Outline.

G. The Master and/or Working Reference should be uniquely identified by lot 

number and expiration date in the filed Outline of Production or Special Outline.

1. The Outline of Production or Special Outline should specify the reference 

(including lot number) used for testing.

2. The Master Reference should be available in sufficient quantities to allow the 

manufacturer and the CVB-L to conduct testing throughout the dating of the 

reference and for an additional period if requalification is anticipated.
Salvaged Portions of Veterinary Services Memorandum No. 800.90 5

V. QUALIFICATION OF A REFERENCE

A. Qualification of a Master Reference for use in in vitro relative potency assays is 

either the initial establishment of a Master Reference or the establishment of a 

new Master Reference to replace an existing reference. The dating of the Master 

Reference is for a defined time period as supported by data and approved by 

APHIS.

B. Master Reference that are unfrozen product references stored similarly to 

product can be directly qualified in animals.

C. Master References that cannot be directly qualified in animals, but require a 

Qualifying Serial for qualification, include:

1. Master References that are frozen product references,

2. Master References that are stabilized and frozen, and 

3. Purified Master References.

D. The immunogenicity of a new and replacement Master Reference should be 

established as prescribed in the applicable 9 CFR Standard Requirement or, for 

agents without codified requirements, in accordance with protocols acceptable to 

APHIS. Alternative immunogenicity proposals for qualifying Master References 

may be considered by APHIS, but should:

1. Include a statistically significant number of animals as would be required for 

initial licensure of a product,

2. Include an assessment of immunogenicity and/or efficacy of a product in the 

host species,

3. Be directly correlated to protection or support label claims, and

4. Be submitted to the firm’s assigned reviewer in the Center for Veterinary 

Biologics-Licensing and Policy Development (CVB-LPD) soon enough to 

allow for comment and approval prior to test initiation.

VI. REQUALIFICATION OF A REFERENCE

A. Requalification of a Master Reference requires demonstration of the 

immunologic stability of a previously qualified Master Reference.

1. The dating of a Master Reference may be extended beyond its expiration 

date by confirming its immunogenicity in a manner acceptable to APHIS.

2. A Master Reference can be requalified multiple times, provided that the 

stability of the Master Reference is shown to be maintained.

B. Requalification of a Master Reference should be done prior to the end of dating.
Salvaged Portions of Veterinary Services Memorandum No. 800.90 6

1. Products tested with expired Master References are not eligible for release.

C. Demonstrating the efficacy of a reference either directly or indirectly is one 

method of confirming immunologic stability.

D. Immunologic methods not requiring vaccination and challenge may demonstrate 

the stability of a reference, provided that the immunologic response was initially 

correlated to protection during a host animal efficacy study.

E. An unfrozen product reference stored similarly to product can be directly 

requalified in animals.

F. Frozen product references, stabilized and frozen references, or purified 

references cannot be directly requalified in animals. The difference in 

composition and/or freeze-thaw cycle precludes direct requalification, as the 

continued correlation to product efficacy cannot be ensured.

G.For references that cannot be directly requalified in animals or where the 

manufacturer wishes to establish a Working Reference different from the Master 

Reference, a Qualifying Serial shall be used in requalification trials.

VII. QUALIFYING SERIAL

A. Qualifying Serials shall be produced in accordance with a filed Outline of 

Production.

B. Dilution of a production serial to produce a Qualifying Serial is allowed, 

provided that after dilution the relative concentration of all the components other 

than the agent being requalified is at release levels per the filed Outline of 

Production.

C. Qualifying Serials should be produced within six (6) months prior to the 

immunogenicity trial, but a serial is acceptable if it was produced within the 

dating period of the product and as specified in the currently filed Outline of 

Production. The use of a recently produced Qualifying Serial ensures that the 

relationship of the reference to the product has not been altered by gradual 

changes in the production method or materials over time.

D. A serial prepared by a method which is different from the method specified in 

the currently filed Outline of Production is not acceptable for use as a Qualifying 

Serial.

E. Qualifying Serials used to requalify a Master Reference or Working Reference 

used in serial release testing shall have a relative potency less than or equal to 1.0 

([1.0) when compared to the Master Reference or Working Reference. A 

minimum of 5 independent replicate assays shall be used to confirm the relative 

potency value of the Qualifying Serial.

F. Qualifying Serials may be sued as Working References, provided that:

1. The Qualifying Serial has a relative potency of 1.0 when compared to the 
Salvaged Portions of Veterinary Services Memorandum No. 800.90 7

Master Reference,

2. The Qualifying Serial is designated as the Working Reference in the filed 

Outline of Production or Special Outline.

G. The dating of Qualifying Serials used as Working Reference shall be:

1. Equal to the dating of the product, or

2. Equal to the dating allowed the Master Reference, if:

a. The relative potency of either the Qualifying Serial or Working 

Reference when compared with the Master Reference is equivalent to 

1.0, 

b. The equivalence of the Qualifying Serial/Master Reference 

relationship is monitored at intervals not less than at mid-dating of the 

Master Reference using the in vitro assay specified in the filed 

Outline of Production or Special Outline, and

c. The monitoring plan is specified in the filed Outline of Product or 

Special Outline.

H. If the Qualifying Serial/Master Reference relationship is shown to be 

nonequivalent, the Qualifying Serial is no longer eligible for use in the test. In 

that event, the Master Reference may be designated as the Working Reference in 

the Outline of Production or Special Outline; or a new Working Reference may 

be qualified.

VIII. STORAGE OF A REFERENCE

A. The storage conditions for the Master or Working Reference used in an in vitro

test for serial release should be stated in the filed Outline of Production or 

Special Outline.

B. Master References that are product references and stored in a manner similar to 

product should not require further dilution or bench-level manipulation prior to 

being used as Working References to test for serial release.

C. Master Reference that are purified antigens and stored in a manner similar to 

product may be diluted up to 1:100 prior to being used as Working References to 

test for serial release. 

D.If stabilizers are used to extend the storage period of a frozen reference that is 

used as a Working Reference in an in vitro test for serial release, the following 

apply:

1. The stabilized frozen reference can be used in the in vitro potency test 

without further sample treatment if:

a. The stabilizer is added prior to freezing the reference,
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b. The stabilizer is added to the reference prior to initiating the 

immunogenicity study,

c. A Qualifying Serial is used in the immunogenicity study, and

d. The relative potency of the stabilized and frozen Master Reference 

and the Qualifying Serial are equivalent when the immunogenicity 

study is initiated. A minimum of five (5) independent replicate assays 

in the in vitro test system shall be used to establish equivalence.

2. If stabilizer is added to the Master Reference after the initiation of the 

immunogenicity study, the in vitro test protocol should provide for similar 

treatment of the test sample. Treatment of the sample should be specified in 

the filed Outline of Production or Special Outline.

3. Mathematical corrections cannot be used to compensate for added stabilizer.

IX. REFERENCE REQUALIFCATION

The purpose of requalifying a Master Reference is to demonstrate its immunogenic 

stability and thereby extend the dating period for use for potency testing. Methods for 

demonstrating immunogenic stability include:

A. Host animal vaccination-challenge studies

1. Host animal protection studies in which a single dilution of the Master 

Reference or Qualifying Serial is administered by the least immunogenic of 

the recommended routes of vaccine administration may be used to extend 

the dating of the Master Reference for a period equal to that allowed for 

product dating.

B. Nonhost animal vaccination-challenge studies

1. Nonhost animal vaccination-challenge studies may be used to demonstrate 

the stability of the Master Reference if the protective immune response in 

nonhost animals is correlated to the protective response in host animals.

C. Host animal serology studies

1. Serological response in the host animal may be used to requalify a Master 

Reference when a relationship between titer and protection has been 

established.

X. TESING ADJUVANTED PRODUCT

A. When both the production serial and the Working Reference contain adjuvant, 

treatment to release bound adjuvant is allowed, provided that:

1. The test serial and the Working Reference receive similar treatment,
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2. The treatment method is specified in the filed Outline of Production or 

Special Outline, and

3. The treatment method was demonstrated to be applicable at the time of an 

immunogenicity or Master Reference requalification study.

B. When adjuvant is present in the test serial but not in the Working Reference, 

treating the test serial to release bound antigen without having to similarly treat 

the Working Reference is allowed if:

1. The serial used in the immunogenicity test was treated, and

2. The treatment method is specified in the filed Outline of Production or 

Special Outline.