The test for detection of extraneous viruses by the fluorescent antibody technique provided in this section shall be conducted when prescribed in an applicable Standard Requirement or in a filed Outline of Production for a product.

(a) Monolayer cultures of cells (monolayers), at least 7 days after the last subculturing, shall be processed and stained with the appropriate antiviral fluorochrome-conjugated antibody as specified in paragraph (b) of this section.

(1) Three groups of one or more monolayers shall be required for each specific virus prescribed in paragraph (b) of this section.

(i) At the time of the last subculturing, one group of test monolayers shall be inoculated with approximately 100-300 FAID50 of the specific virus being tested for as positive controls.

(ii) One group of monolayers shall be the “material under test.”

(iii) One group of monolayers, that are of the same type of cells as the test monolayers and that have been tested as prescribed in §§113.51 or 113.52 (whichever is applicable), shall be prepared as negative controls.

(2) Each group of monolayers shall have a total area of at least 6 cm2.

(3) Positive control monolayers may be fixed (processed so as to arrest growth and assure attachment of the monolayer to the surface of the vessel in which they are grown) before 7 days after subculturing if fluorescence is enhanced by doing so, Provided, That a monolayer of the material under test is also fixed at the same time as the positive control and a monolayer of the material under test is also fixed at least seven days after subculturing. Monolayers that are fixed before 7 days after subculturing shall be stained at the same time as the test monolayers and negative controls fixed at least 7 days after subculturing.

(b) The antiviral fluorochrome-conjugated antibodies to be used shall depend on the type of cells required to be tested for extraneous viruses as specified in an applicable Standard Requirement or in a filed Outline of Production. Antiviral fluorochrome-conjugated antibodies specific for the extraneous viruses shall be applied to each respective type of cell in accordance with the following list. Under certain circumstances, additional tests may need to be conducted, as determined by the Administrator. When a specific antiviral fluorochrome-conjugated antibody is used in testing for the listed extraneous viruses specified in more than one cell type, it need only be applied to the most susceptible cell type.

(1) All cells shall be tested for:

(i) Bovine virus diarrhea virus;

(ii) Reovirus; and

(iii) Rabies virus.

(2) Bovine, caprine, and ovine cells shall, in addition, be tested for:

(i) Bluetongue virus;

(ii) Bovine adenoviruses;

(iii) Bovine parvovirus; and

(iv) Bovine respiratory syncytial virus.

(3) Canine cells shall, in addition, be tested for:

(i) Canine coronavirus;

(ii) Canine distemper virus; and

(iii) Canine parvovirus.

(4) Equine cells shall, in addition, be tested for:

(i) Equine herpesvirus; and

(ii) Equine viral arteritis virus.

(5) Feline cells shall, in addition, be tested for:

(i) Feline infectious peritonitis virus; and

(ii) Feline panleukopenia virus.

(6) Porcine cells shall, in addition, be tested for:

(i) Porcine adenovirus;

(ii) Porcine parvovirus;

(iii) transmissible gastroenteritis virus; and

(iv) Porcine hemagglutinating encephalitis virus.

(7) Firms that do not have rabies virus on premises either for research or production purposes are exempt from having to produce positive rabies virus control monolayers. Fixed positive rabies virus control monolayers will be provided by the National Veterinary Services Laboratories.

(c) After staining, each group of monolayers shall be examined for the presence of specific fluorescence attributable to the presence of extraneous viruses.

(1) If the material under test shows any evidence of specific viral fluorescence, it is unsatisfactory and may not be used; Provided, That, if specific fluorescence attributable to the virus being tested for is absent in the positive control monolayers, the test is a No Test and may be repeated.

(2) If the fluorescence of the monolayers inoculated with the specific virus as positive controls is equivocal, or if the negative monolayers show equivocal fluorescence indicating possible viral contamination, or both, the test shall be declared a No Test, and may be repeated; Provided, That, if the test is not repeated, the material under test shall be regarded as unsatisfactory for use in the production of biologics.

[60 FR 24548, May 9, 1995]