- Email: [email protected]
Licensing veterinary diagnostic test kits in the United States
142 CLINICAL I M M U N O L O G Y Newsletter
Vol. 13, No. 11, 1993
L i c e n s i n g V e t e r i n a r y D i a g n o s t i c Test Kits in the U n i t e d States Cyril G. G a y a n d A l b e r t P. M o r g a n
UnitedStatesDepartmentof Agriculture(USDA), Animaland PlantHealthInspectionService(APHIS),Biotechnology,Biologics,and EnvironmentalProtection(BBEP),VeterinaryBiologics,Hyattsville,Maryland s infectious diseases continue to challenge the veterinary profession, the need for commercially available diagnostic test kits is increasing. Few diagnostic laboratories provide veterinary services and test results are often too late and too expensive. As preventive medicine continues to play an important role in veterinary medicine, clinicians are finding diagnostic kits to be a rational approach to diagnosis, prognosis, and clinical management. Recent technological innovations and advances have conlributed to the development of new diagnostic test kits that are simple, fast, and accurate. As a result, there has been an upsurge of interest in licensing these products. As illustrated in Figure 1, there are currently 148 diagnostic test kits licensed in the U.S. A total of 27 firms have been licensed to manufacture these products. Of these, 12 firms manufactare diagnostic test kits with components that are products of biotechnology. These include monoclonal antibodies used in enzyme-linked immunosorbent assays (E,LISA) and cloned DNA and RNA fragments used for molecular probes and the polymerase chain reaction (PCR). Biotechnology-derived diagnostic test kits are classifted as Category I Veterinary Biologics? To date, 42 biotechnology-derived diagnostic test kits have been licensed. Examples of these diagnostic test kits are provided in Table 1. The United States Department of Agriculture (USDA) is responsible for establishing licensing and test requirements for veterinary diagnostic test kits. The goal is to ensure that these products are specific, sensitive, stable, and give reproducible resuits. Only complete kits are licensed, i.e., kits that include all reagents and instructions required for use of the product in making a diagnosis. The following outlines the applicable regulations and requirements for licensing veterinary diagnostic
A
0197-I859/93/$0.00+ 6.00
test kits, including molecular diagnostics. Applicable Regulations The USDA final policy statement for regulating biotechnology products was published in the Federal Register June 26,
A s preventive medicine continues to play an important role in veterinary medicine, clinicians are finding diagnostic kits to be a rational approach to diagnosis, prognosis, and clinical management. Recent technological innovations and advances have contributed to the development o f new diagnostic test kits that are simple,fast, and accurate.
1986. 2 This document provided the USDA portion of the coordinated framework for regulating biotechnology in the U.S. The policy stated that agriculture products developed by biotechnology do not differ fundamentally from conventional products and that the existing regulatory framework is adequate to regulate biotechnology. The authority for the regulation of veterinary biological products in the U.S. is provided in the Virus-Serum-Toxin Act (VSTA) of 1913) The VSTA was enacted by congress to prevent the importation and interstate shipment of worthless, contaminated, dangerous, or harmful veterinary biological products. The VSTA was amended by the Food Security Act of 1985 © 1993ElsevierSciencePublishingCo., Inc.
to include all products shipped into, within, or from the U.S. The USDA is charged with promulgating regulations and procedures consistent with the Act to ensure that veterinary biological products are pure, safe, potent, and efficacious. Veterinary Biologics (VB), Biotechnology, Biologics, and Environmental Protection (BBEP), Animal and Plant Health Inspection Service (APHIS), administers the pursuant regulations, Title 9, Code of Federal Regulations (9 CFR), Parts 101 to 118. 5 Parts 101-112 contain administrative details and definitions, Parts 113-114 contain Standard and Production Requirements, and Parts 115-118 cover facility inspections and record-keeping. Currently, 9 CFR Part 113 only prescribes general requirements for live and killed bacterial and virus vaccines, and biological products of animal blood origin. Generally, diagnostic test kits are subject to the same or similar licensing requirements. The definition of a veterinary biological products is provided in Part 101.2: [veterinary biological products] means all viruses, serums, toxins, and analogous products such as diagnostics, antitoxins, vaccines, live microorganism, killed microorganisms and antigenic or immunizing components of microorganisms intended for use in the diagnosis, treatment, or prevention of diseases of animals. A working defmition of a veterinary diagnostic test kit is "the ingredients, reagents and serums, probes and/or other components that may be included as part of a kit or other device used in the diagnosis of disease, or for use in making a determination of susceptibility to and/or exposure to potential disease causing agents in animals." The Veterinary Biologics Program The Veterinary Biologics Program comprises three main units that coordinate their activities and functions. Program
CLINICAL IMMUNOLOGY Newsletter 143
Vol. 13, No. 11, 1993
functions include staff, field, and laboratory responsibilities. Staff Responsibilities Veterinary Biologics, a staff unit, issues licenses both for production facilities and for products, and issues import permits as well. It establishes licensing requirements regarding testing methods, lxoduction methods, and product labels. It also develops general policy regarding veterinary biologics regulation.
Product Licenses 60 4 0
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20 O 0
80 60 40
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Field Responsibilities Veterinary Biologics Held Operations (VBFO), a line unit, carries out late- and postlicense inspections of the production facilities and processes. After aplxoval of test results, it releases serials 0ors) of product for disaibution in the marketplace. It also re, sponds to consumer complaints and coordinates investigations of sus~ted violations. Laboratory Responsibilities National Veterinary Services Laboratories, the program laboratory unit, tests products prior to licensure and tests randomly selected serials (lots) of licensed products to monitor licensees' quality control. It provides producers and others with test references and reagents, trains personnel from other laboratories, and develops new testing methods.
o
80 81
=
=
82 83 84 85 86 87 88 89 90 91
92 93
F i s c a l Year Jim Licensed [--] Biotechnology Derived Figure 1. Numberof veterinarydiagnostictest kits licensedin the U.S.
preparation of the product. The Outline of Production should be prepared and submitted in accordance with 9 CFR 114.8 and 9 CFR 114.9.(t"). Summary Information Format Summary Information Formats specify the relevant information to submit to APHIS
for new product license applications? Four Summary Infotfaation Formats are available, each representing a different category of veterinary biologics. The Summary Information Format that is applicable to molecular diagnostics is provided in Table 2. The molecular properties of the master seed microorganism are identified, as well
Licensing Requirements
TABLE 1. EXAMPLF~OF LICENSEDVETERINARYDIAGNOSTICIEST KITS
Licensing is initiated with the filing of an establishment and product license application (APHIS Forms 2001 and 2003). Supporting materials for a product license application include an outline of production, a completed Summary Information Format (for moleeular diagnostics), sketches and final labels, and supporting d~ta to demonstrate the purity, safety, potency, and efficacy of the l~oduct.
Product
Code no.
Licensed finn
Estab. no. Date licensed
Feline Leukemia Vires Antibody Test Kit
5028.00
Cambridge BioScience Corporation
317
October 30, 1985
Equine Infectious Anemia Antibody Test Kit, ELISA
5515.00
Bio-Vac Laboratories, Inc.
272
November 20,1987
Feline Immtmodeficiency Vires Antibody Test Kit
5036.00
IDEXX Laboratories, Inc.
313
February 26, 1988
Mycobacterium Paratuborculmis DNA Test Kit
5066.00
IDEXX Laboratories, Inc.
313
September 12, 1990
Mycoplasma Galliseptictan DNA Test Kit
5A70.00
IDEXX Laboratories, Inc.
313
July 21, 1992
Mycobactemim Paratuberculosis Gamma Interferon Test Kit
51364.00
IDEXX Laboratories, Inc.
313
September 25, 1992
Agdia, Inc.
369
March 10, 1993
Outline of Production The outline of production is the detailed protocol of methods to be followed in the
Pseudorabies V i m gpX Antibody 5111.01 Test Kit
O 1993 Elsevier Science Publishing Co., Inc.
019%1859/93/$0.00 + 6.00
144 CLINICAL I M M U N O L O G Y Newsletter
Vol. 13, Nol ! - ~ , 1993
I
TABLE 2. SUMMARY INFORMATION FORMAT FOR CATEGORY I VETERINARY BIOLOGICS Characterization of the recombinant organism
Purity
A. Molecular properties I.
Recipient characterization (a) Parenteral organism CO) Description of the recipient organism prior to receiving the deletion(s) and/or donor gene (c) Genetic modifications used to produce the recipient organism form the parenteral organism
2.
Characterization of the deletion(s), where applicable (a) Deleted gene(s) CO) Proposed phenotypic effect of the deletion(s) upon the recipient organism
3.
Characterization of the donor gene(s), where applicable (a) Donor organism (b) Donor gene(s), including any inserted marker genes
4.
Construction of the recombinant organism (a) Summary of the construction process Co) Intermediate cloning vector(s) (c) Procedure for introducing the genetic modification(s) to the recipient (d) Procedure for introducing the genetic modification(s) to the donor genes (e) Screening methods and protocols for the identification and purification of the recombinant organism
5.
Molecular characterization of the Master Seed (a) Master Seed designation Co) Method(s) and protocols used to establish identification of the Master Seed (c) Stability of the Master Seed organism at the n and n+5 (highest passage level)
B.
Biological properties 1.
requirements applied to vaccines and bacterins.
Master Seed
Diagnostic products are required to be produced with clean seeds, substrates, and ingredients. The requirements imposed on seeds, substrates, and ingredients are intended to ensure that all products are free from extraneous bacteria, fungi, mycoplasma, and viruses. Freedom from contaminating microorganisms is demonstrated by testing seeds, substrates, and ingredients in accordance with the following Standard Requirements, where applicable: detection of extraneous viable bacteria and fungi in live vaccines (9 CFR 113.27); detection of Salmonella contamination (113.30); detection of avian lymphoid leukosis (9 CFR 113.31); detection of hemagglutinating viruses (9 CFR 113.34); ingredients of biological products (9 CFR 113.50); requirements for primary cells used for production of biologics (9 CFR 113.51); requirements for cell lines used for production of biologics (9 CFR 113.52); requirements for ingredients of animal origin used for production of biologics (9 CFR 113.53); detection of extraneous agents in master seed virus (9 CFR 113.55).
(a) Purity Co) Inactivation procedures
as those of the recipient microorganism, and any deleted or donor genes. A detailed description of the methods used to develop the master seed microorganism is provided, as would be acceptable in a "materials and methods" section of a refereed scientific journal. The construction process should be clearly described. The genetic modifications used to construct the master seed microorganisms are identified, including deleted genes, structural donor genes, regulatory elements of the donor genes, intermediate cloning vectors, etc. The purity and inactivation of the master seed must be confirmed. Inactivation procedures and supporting data are provided. Purity requirements for master illn
0197-1859/93/$0.00 + 6.00
Safety
seeds are outlined below. Sketches and Final Labels Firms must submit for review sketches and proposed final labels. The label indications and all claims that are made on the label must be supported with appropriate data filed with the USDA. The regulations governing packaging and labeling appear in 9 CFR 112. Supporting Data To qualify for licensure, all biological products must be shown to be pure, safe, potent, and efficacious. Diagnostic test kits are subject to the same or similar licensing
ii
i
© 1993 Elsevier Science Publishing Co., Inc.
The reagents, substrates, and ingredients that make up a diagnostic test kit do not in general pose any safety risks. Notwithstanding, the safety of these materials is assessed. Primary consideration is given to laboratory personnel working with recombinant master seeds during the testing phase. APHIS conducts risk assessments and identifies appropriate biosafety containment levels prior to testing recombinant master seeds at NVSL. Potency Prior to release, each serial of production is tested by the manufacturer for potency using criteria approved by APHIS; e.g., test controls, reference serum, serial release panels, The criteria for testing potency are documented in Section V, Part C, of the outline of production. Results arc
C L I N I C A L I M M U N O L O G Y Newsletter 145
Vol. 13, No. 11, 1993
submitted to VBFO, which approves the release of all serials based on the manufacturer's satisfactory tests. Manufacturers must send samples of all serials to NVSL where they are held beyond dating. Within 3 days of receipt, serials are randomly selected and tested for potency. Serials not selected are eligible for release by VBFO. Serials that are selected for testing at NVSL are not released until after NVSL has confirmed that the diagnostic test kit performs satisfactorily. Efficacy Diagnostic products must with reasonable ceaainty yield the results intended when used as recommended or suggested by the product label. The primary licensing considerations are sensitivity, specificity, and reproducibility. Sensitivity is expressed as the percentage of positive results in animals known to have the disease and is usually established by comparison with a "gold standard," if one exists, or other diagnostic procedures and assay methods (e.g., virus serum neutralization, complement fixation). The minimum detectable level expressed in titer of antibody or nanograms of antigen should be established. Test kits for the detection of antigen should be tested against related antigens to detect false-positive reactions. Test kits should have at least one positive reference standard that has been characterized. Specificity is expressed as the percentage of negative results in animals known to be disease free, and is usually established by evaluating kit performance against serums from animals vaccinated with commonly used immunizing agents or antigens to detect cross-reactivity. Although molecular probes should provide the ultimate specificity and sensitivity results, problems do exist. For example, cross-reactions may occur if the selected gene sequences are shared with other agents. In the case of the Mycobacterium Paratuberculosis DNA Test Kit licensed by Idexx Laboratories, Inc.,7 sensitivity was evaluated with fecal culture positive samples and with normal fecal samples "spiked" with dilutions of Mycobacterium paratuberculosis. Specificity was evalu-
ated using pure cultures of other Mycobacteria sp. and fecal samples from animals confirmed free of Mycobacteriumparatuberculosis. The mycobacterial cultures assayed consisted of 13 serotypes of M. avium, M. bovis isolates, M. flavescens, M.
and achieving "official" status may vary between 6 months and 1 year. Diagnostic kits used in the brucellosis eradication and pseudorabies control programs are exampies. Conclusion
New recombinant vaccines with gene additions and~or deletions are now being matched with "companion" diagnostic test kits that can differentiate between vaccinated and infected animals. The use of PCR now provides a sensitive method for detecting DNA from infectious microorganisms such as M. paratuberculosis.
gondoneae, M. M. kansii, M. marinum, M. phlei, M. smegmatis, and M. xenopi. Reproducibility is a measurement applied to quantitative kits. Usually, multiple measurements are taken on reference serum panel to establish a coefficient of variation (CV). The CV mast be within acceptable limits for the planned application of the kit. Manufacturers are asked to provide kits to several laboratories for evaluation. Participating laboratories are asked to evaluate the kit in comparison to the diagnostic procedure routinely used in the laboratory. These comparative data are also used in determining the suitability of the kit for licensing. Diagnostic test kits intended for use in Cooperative State/Federal/Industry Animal Disease Control and Eradication Programs are subject to additional evaluation as directed by APHIS. Field studies are coordinated with the American Association of Veterinary Laboratory Diagnosticians, the United States Animal Health Association, and NVSL. The time between licensing
© 1993 Elsevier Science Publishing Co., Inc.
A tremendous interest in licensing veterinary diagnostic test kits has developed in the last decade. Technological innovations and advances have provided new products that are simple, rapid, and accurate. New recombinant vaccines with gene additions and/or deletions are now being matched with "companion" diagnostic test kits that can differentiate between vaccinated and infected animals. The use of PCR now provides a sensitive method for detecting DNA from infectious microorganisms such as M. paratuberculosis. Licensing requirements ensure the specificity and sensitivity of veterinary diagnostic test kits. Diagnostic test kits are subject to the same requirements applied to veterinary vaccines. Requirements are based on the best science. A Summary Information Format is available for molecular diagnostics. Several nucleic acid probe test kits have met USDA requirements and are now commercially available. ClN References 1.
2.
3. 4. 5.
6.
7.
Gay CG: Current USDA procedures for licensing biotechnology-dedved veterinary biologicals. Dev Biol Stand 79:65-74, 1992. Executive Office of the President, Office of Science and Technology Policy: Coordinated framework for regulation of biotechnology. Federal Register, 51, 23302, 1986. U.S. Government Printing Office, Washington, DC. 21 United States Code: §§ 151-158, 1982. Public Law No. 99-198:99 Slat. 1354, 1985. Code of Federal Regulations: Title 9, Parts 1 to 199. U.S. Government Printing Office, Washington, DC. Gay CG, Roth HJ: Confirming the safety characteristics of recombinant vectors used in veterinary medicine: a regulatory perspective. Dev Biol Stand, in press. Vary PH, Andersen PR, Green E, et al: Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne's Disease. J. Clin. Microbiol. 28:933--937, 1990.
0197-1859D3/$0.00 + 6.00
Vol. 13, No. 11, 1993
L i c e n s i n g V e t e r i n a r y D i a g n o s t i c Test Kits in the U n i t e d States Cyril G. G a y a n d A l b e r t P. M o r g a n
UnitedStatesDepartmentof Agriculture(USDA), Animaland PlantHealthInspectionService(APHIS),Biotechnology,Biologics,and EnvironmentalProtection(BBEP),VeterinaryBiologics,Hyattsville,Maryland s infectious diseases continue to challenge the veterinary profession, the need for commercially available diagnostic test kits is increasing. Few diagnostic laboratories provide veterinary services and test results are often too late and too expensive. As preventive medicine continues to play an important role in veterinary medicine, clinicians are finding diagnostic kits to be a rational approach to diagnosis, prognosis, and clinical management. Recent technological innovations and advances have conlributed to the development of new diagnostic test kits that are simple, fast, and accurate. As a result, there has been an upsurge of interest in licensing these products. As illustrated in Figure 1, there are currently 148 diagnostic test kits licensed in the U.S. A total of 27 firms have been licensed to manufacture these products. Of these, 12 firms manufactare diagnostic test kits with components that are products of biotechnology. These include monoclonal antibodies used in enzyme-linked immunosorbent assays (E,LISA) and cloned DNA and RNA fragments used for molecular probes and the polymerase chain reaction (PCR). Biotechnology-derived diagnostic test kits are classifted as Category I Veterinary Biologics? To date, 42 biotechnology-derived diagnostic test kits have been licensed. Examples of these diagnostic test kits are provided in Table 1. The United States Department of Agriculture (USDA) is responsible for establishing licensing and test requirements for veterinary diagnostic test kits. The goal is to ensure that these products are specific, sensitive, stable, and give reproducible resuits. Only complete kits are licensed, i.e., kits that include all reagents and instructions required for use of the product in making a diagnosis. The following outlines the applicable regulations and requirements for licensing veterinary diagnostic
A
0197-I859/93/$0.00+ 6.00
test kits, including molecular diagnostics. Applicable Regulations The USDA final policy statement for regulating biotechnology products was published in the Federal Register June 26,
A s preventive medicine continues to play an important role in veterinary medicine, clinicians are finding diagnostic kits to be a rational approach to diagnosis, prognosis, and clinical management. Recent technological innovations and advances have contributed to the development o f new diagnostic test kits that are simple,fast, and accurate.
1986. 2 This document provided the USDA portion of the coordinated framework for regulating biotechnology in the U.S. The policy stated that agriculture products developed by biotechnology do not differ fundamentally from conventional products and that the existing regulatory framework is adequate to regulate biotechnology. The authority for the regulation of veterinary biological products in the U.S. is provided in the Virus-Serum-Toxin Act (VSTA) of 1913) The VSTA was enacted by congress to prevent the importation and interstate shipment of worthless, contaminated, dangerous, or harmful veterinary biological products. The VSTA was amended by the Food Security Act of 1985 © 1993ElsevierSciencePublishingCo., Inc.
to include all products shipped into, within, or from the U.S. The USDA is charged with promulgating regulations and procedures consistent with the Act to ensure that veterinary biological products are pure, safe, potent, and efficacious. Veterinary Biologics (VB), Biotechnology, Biologics, and Environmental Protection (BBEP), Animal and Plant Health Inspection Service (APHIS), administers the pursuant regulations, Title 9, Code of Federal Regulations (9 CFR), Parts 101 to 118. 5 Parts 101-112 contain administrative details and definitions, Parts 113-114 contain Standard and Production Requirements, and Parts 115-118 cover facility inspections and record-keeping. Currently, 9 CFR Part 113 only prescribes general requirements for live and killed bacterial and virus vaccines, and biological products of animal blood origin. Generally, diagnostic test kits are subject to the same or similar licensing requirements. The definition of a veterinary biological products is provided in Part 101.2: [veterinary biological products] means all viruses, serums, toxins, and analogous products such as diagnostics, antitoxins, vaccines, live microorganism, killed microorganisms and antigenic or immunizing components of microorganisms intended for use in the diagnosis, treatment, or prevention of diseases of animals. A working defmition of a veterinary diagnostic test kit is "the ingredients, reagents and serums, probes and/or other components that may be included as part of a kit or other device used in the diagnosis of disease, or for use in making a determination of susceptibility to and/or exposure to potential disease causing agents in animals." The Veterinary Biologics Program The Veterinary Biologics Program comprises three main units that coordinate their activities and functions. Program
CLINICAL IMMUNOLOGY Newsletter 143
Vol. 13, No. 11, 1993
functions include staff, field, and laboratory responsibilities. Staff Responsibilities Veterinary Biologics, a staff unit, issues licenses both for production facilities and for products, and issues import permits as well. It establishes licensing requirements regarding testing methods, lxoduction methods, and product labels. It also develops general policy regarding veterinary biologics regulation.
Product Licenses 60 4 0
. . . . . . . . . . . . . . . . . . .
20 O 0
80 60 40
.
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Field Responsibilities Veterinary Biologics Held Operations (VBFO), a line unit, carries out late- and postlicense inspections of the production facilities and processes. After aplxoval of test results, it releases serials 0ors) of product for disaibution in the marketplace. It also re, sponds to consumer complaints and coordinates investigations of sus~ted violations. Laboratory Responsibilities National Veterinary Services Laboratories, the program laboratory unit, tests products prior to licensure and tests randomly selected serials (lots) of licensed products to monitor licensees' quality control. It provides producers and others with test references and reagents, trains personnel from other laboratories, and develops new testing methods.
o
80 81
=
=
82 83 84 85 86 87 88 89 90 91
92 93
F i s c a l Year Jim Licensed [--] Biotechnology Derived Figure 1. Numberof veterinarydiagnostictest kits licensedin the U.S.
preparation of the product. The Outline of Production should be prepared and submitted in accordance with 9 CFR 114.8 and 9 CFR 114.9.(t"). Summary Information Format Summary Information Formats specify the relevant information to submit to APHIS
for new product license applications? Four Summary Infotfaation Formats are available, each representing a different category of veterinary biologics. The Summary Information Format that is applicable to molecular diagnostics is provided in Table 2. The molecular properties of the master seed microorganism are identified, as well
Licensing Requirements
TABLE 1. EXAMPLF~OF LICENSEDVETERINARYDIAGNOSTICIEST KITS
Licensing is initiated with the filing of an establishment and product license application (APHIS Forms 2001 and 2003). Supporting materials for a product license application include an outline of production, a completed Summary Information Format (for moleeular diagnostics), sketches and final labels, and supporting d~ta to demonstrate the purity, safety, potency, and efficacy of the l~oduct.
Product
Code no.
Licensed finn
Estab. no. Date licensed
Feline Leukemia Vires Antibody Test Kit
5028.00
Cambridge BioScience Corporation
317
October 30, 1985
Equine Infectious Anemia Antibody Test Kit, ELISA
5515.00
Bio-Vac Laboratories, Inc.
272
November 20,1987
Feline Immtmodeficiency Vires Antibody Test Kit
5036.00
IDEXX Laboratories, Inc.
313
February 26, 1988
Mycobacterium Paratuborculmis DNA Test Kit
5066.00
IDEXX Laboratories, Inc.
313
September 12, 1990
Mycoplasma Galliseptictan DNA Test Kit
5A70.00
IDEXX Laboratories, Inc.
313
July 21, 1992
Mycobactemim Paratuberculosis Gamma Interferon Test Kit
51364.00
IDEXX Laboratories, Inc.
313
September 25, 1992
Agdia, Inc.
369
March 10, 1993
Outline of Production The outline of production is the detailed protocol of methods to be followed in the
Pseudorabies V i m gpX Antibody 5111.01 Test Kit
O 1993 Elsevier Science Publishing Co., Inc.
019%1859/93/$0.00 + 6.00
144 CLINICAL I M M U N O L O G Y Newsletter
Vol. 13, Nol ! - ~ , 1993
I
TABLE 2. SUMMARY INFORMATION FORMAT FOR CATEGORY I VETERINARY BIOLOGICS Characterization of the recombinant organism
Purity
A. Molecular properties I.
Recipient characterization (a) Parenteral organism CO) Description of the recipient organism prior to receiving the deletion(s) and/or donor gene (c) Genetic modifications used to produce the recipient organism form the parenteral organism
2.
Characterization of the deletion(s), where applicable (a) Deleted gene(s) CO) Proposed phenotypic effect of the deletion(s) upon the recipient organism
3.
Characterization of the donor gene(s), where applicable (a) Donor organism (b) Donor gene(s), including any inserted marker genes
4.
Construction of the recombinant organism (a) Summary of the construction process Co) Intermediate cloning vector(s) (c) Procedure for introducing the genetic modification(s) to the recipient (d) Procedure for introducing the genetic modification(s) to the donor genes (e) Screening methods and protocols for the identification and purification of the recombinant organism
5.
Molecular characterization of the Master Seed (a) Master Seed designation Co) Method(s) and protocols used to establish identification of the Master Seed (c) Stability of the Master Seed organism at the n and n+5 (highest passage level)
B.
Biological properties 1.
requirements applied to vaccines and bacterins.
Master Seed
Diagnostic products are required to be produced with clean seeds, substrates, and ingredients. The requirements imposed on seeds, substrates, and ingredients are intended to ensure that all products are free from extraneous bacteria, fungi, mycoplasma, and viruses. Freedom from contaminating microorganisms is demonstrated by testing seeds, substrates, and ingredients in accordance with the following Standard Requirements, where applicable: detection of extraneous viable bacteria and fungi in live vaccines (9 CFR 113.27); detection of Salmonella contamination (113.30); detection of avian lymphoid leukosis (9 CFR 113.31); detection of hemagglutinating viruses (9 CFR 113.34); ingredients of biological products (9 CFR 113.50); requirements for primary cells used for production of biologics (9 CFR 113.51); requirements for cell lines used for production of biologics (9 CFR 113.52); requirements for ingredients of animal origin used for production of biologics (9 CFR 113.53); detection of extraneous agents in master seed virus (9 CFR 113.55).
(a) Purity Co) Inactivation procedures
as those of the recipient microorganism, and any deleted or donor genes. A detailed description of the methods used to develop the master seed microorganism is provided, as would be acceptable in a "materials and methods" section of a refereed scientific journal. The construction process should be clearly described. The genetic modifications used to construct the master seed microorganisms are identified, including deleted genes, structural donor genes, regulatory elements of the donor genes, intermediate cloning vectors, etc. The purity and inactivation of the master seed must be confirmed. Inactivation procedures and supporting data are provided. Purity requirements for master illn
0197-1859/93/$0.00 + 6.00
Safety
seeds are outlined below. Sketches and Final Labels Firms must submit for review sketches and proposed final labels. The label indications and all claims that are made on the label must be supported with appropriate data filed with the USDA. The regulations governing packaging and labeling appear in 9 CFR 112. Supporting Data To qualify for licensure, all biological products must be shown to be pure, safe, potent, and efficacious. Diagnostic test kits are subject to the same or similar licensing
ii
i
© 1993 Elsevier Science Publishing Co., Inc.
The reagents, substrates, and ingredients that make up a diagnostic test kit do not in general pose any safety risks. Notwithstanding, the safety of these materials is assessed. Primary consideration is given to laboratory personnel working with recombinant master seeds during the testing phase. APHIS conducts risk assessments and identifies appropriate biosafety containment levels prior to testing recombinant master seeds at NVSL. Potency Prior to release, each serial of production is tested by the manufacturer for potency using criteria approved by APHIS; e.g., test controls, reference serum, serial release panels, The criteria for testing potency are documented in Section V, Part C, of the outline of production. Results arc
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submitted to VBFO, which approves the release of all serials based on the manufacturer's satisfactory tests. Manufacturers must send samples of all serials to NVSL where they are held beyond dating. Within 3 days of receipt, serials are randomly selected and tested for potency. Serials not selected are eligible for release by VBFO. Serials that are selected for testing at NVSL are not released until after NVSL has confirmed that the diagnostic test kit performs satisfactorily. Efficacy Diagnostic products must with reasonable ceaainty yield the results intended when used as recommended or suggested by the product label. The primary licensing considerations are sensitivity, specificity, and reproducibility. Sensitivity is expressed as the percentage of positive results in animals known to have the disease and is usually established by comparison with a "gold standard," if one exists, or other diagnostic procedures and assay methods (e.g., virus serum neutralization, complement fixation). The minimum detectable level expressed in titer of antibody or nanograms of antigen should be established. Test kits for the detection of antigen should be tested against related antigens to detect false-positive reactions. Test kits should have at least one positive reference standard that has been characterized. Specificity is expressed as the percentage of negative results in animals known to be disease free, and is usually established by evaluating kit performance against serums from animals vaccinated with commonly used immunizing agents or antigens to detect cross-reactivity. Although molecular probes should provide the ultimate specificity and sensitivity results, problems do exist. For example, cross-reactions may occur if the selected gene sequences are shared with other agents. In the case of the Mycobacterium Paratuberculosis DNA Test Kit licensed by Idexx Laboratories, Inc.,7 sensitivity was evaluated with fecal culture positive samples and with normal fecal samples "spiked" with dilutions of Mycobacterium paratuberculosis. Specificity was evalu-
ated using pure cultures of other Mycobacteria sp. and fecal samples from animals confirmed free of Mycobacteriumparatuberculosis. The mycobacterial cultures assayed consisted of 13 serotypes of M. avium, M. bovis isolates, M. flavescens, M.
and achieving "official" status may vary between 6 months and 1 year. Diagnostic kits used in the brucellosis eradication and pseudorabies control programs are exampies. Conclusion
New recombinant vaccines with gene additions and~or deletions are now being matched with "companion" diagnostic test kits that can differentiate between vaccinated and infected animals. The use of PCR now provides a sensitive method for detecting DNA from infectious microorganisms such as M. paratuberculosis.
gondoneae, M. M. kansii, M. marinum, M. phlei, M. smegmatis, and M. xenopi. Reproducibility is a measurement applied to quantitative kits. Usually, multiple measurements are taken on reference serum panel to establish a coefficient of variation (CV). The CV mast be within acceptable limits for the planned application of the kit. Manufacturers are asked to provide kits to several laboratories for evaluation. Participating laboratories are asked to evaluate the kit in comparison to the diagnostic procedure routinely used in the laboratory. These comparative data are also used in determining the suitability of the kit for licensing. Diagnostic test kits intended for use in Cooperative State/Federal/Industry Animal Disease Control and Eradication Programs are subject to additional evaluation as directed by APHIS. Field studies are coordinated with the American Association of Veterinary Laboratory Diagnosticians, the United States Animal Health Association, and NVSL. The time between licensing
© 1993 Elsevier Science Publishing Co., Inc.
A tremendous interest in licensing veterinary diagnostic test kits has developed in the last decade. Technological innovations and advances have provided new products that are simple, rapid, and accurate. New recombinant vaccines with gene additions and/or deletions are now being matched with "companion" diagnostic test kits that can differentiate between vaccinated and infected animals. The use of PCR now provides a sensitive method for detecting DNA from infectious microorganisms such as M. paratuberculosis. Licensing requirements ensure the specificity and sensitivity of veterinary diagnostic test kits. Diagnostic test kits are subject to the same requirements applied to veterinary vaccines. Requirements are based on the best science. A Summary Information Format is available for molecular diagnostics. Several nucleic acid probe test kits have met USDA requirements and are now commercially available. ClN References 1.
2.
3. 4. 5.
6.
7.
Gay CG: Current USDA procedures for licensing biotechnology-dedved veterinary biologicals. Dev Biol Stand 79:65-74, 1992. Executive Office of the President, Office of Science and Technology Policy: Coordinated framework for regulation of biotechnology. Federal Register, 51, 23302, 1986. U.S. Government Printing Office, Washington, DC. 21 United States Code: §§ 151-158, 1982. Public Law No. 99-198:99 Slat. 1354, 1985. Code of Federal Regulations: Title 9, Parts 1 to 199. U.S. Government Printing Office, Washington, DC. Gay CG, Roth HJ: Confirming the safety characteristics of recombinant vectors used in veterinary medicine: a regulatory perspective. Dev Biol Stand, in press. Vary PH, Andersen PR, Green E, et al: Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne's Disease. J. Clin. Microbiol. 28:933--937, 1990.
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