The IHC Test Variables:

• IHC has been the primary confirmatory test for 

USDA’s BSE surveillance program and is recognized 

by the World Organization for Animal Health, or OIE.

• IHC allows scientists to determine if a sample is 

positive for BSE in two distinct ways:

1.) A staining technique (presence of abnormal 

prion protein) that uses antibodies to detect 

abnormal prion protein in the brain.

2.) A visual examination to determine whether there 

are lesions (holes or “spongy” appearances) 

present in the brain.

• Several variables could yield conflicting results:

o IHC is not a standardized, commercially 

available test. It involves variables, including 

several options in types of antibodies and other 

reactive agents.The sensitivity of any given test 

is influenced by those variables.

o If the level of infectivity in the animal is extremely 

low, the abnormal prion in the brain will be 

minimal and therefore more difficult to detect.

o Variations in the conditions under which the 

staining process is performed, such as 

chemicals and reactive agents used, 

temperature and length of antibody exposure, 

can also cause the test to yield different results.

Testing History on This Animal:

• In November 2004, a sample from this animal 

returned inconclusive for BSE on a Biorad screening 

test.

• The sample was subjected to an IHC confirmatory 

test, which returned negative.

• USDA scientists also ran an additional, experimental 

IHC “rapid” tissue fixation test for academic purposes,

which can be conducted more quickly than the IHC 

confirmatory test and is therefore of interest to the 

scientific community, but it has not been approved 

internationally.

• While some abnormalities were noted in the 

experimental IHC test results, because the test was 

not a validated procedure, and because the two 

approved IHC tests came back negative, the results 

were not considered to be of regulatory significance 

and therefore were not reported beyond the 

laboratory.

• A Western blot test conducted the week of 

June 5, 2005, returned positive for BSE.

• An additional IHC confirmatory test conducted the 

week of June 13, 2005, by USDA scientists utilizing 

different antibodies from the November 2004 test, 

confirmed this case as weakly positive for BSE.

• The Veterinary Laboratories Agency in Weybridge, 

England, conducted a series of diagnostic tests 

including an IHC, using different antibodies from 

those used by USDA in November 2004, which 

returned positive results for BSE.

• Experts from the Weybridge lab confirmed the 

accuracy of the results of USDA’s November 

confirmatory IHC test, concurring that the case could 

not have been confirmed on the basis of this sample.

• Weybridge experts also examined the November 

experimental IHC test and interpreted the results to 

be positive.

Potential Causes of Conflicting Results:

• USDA scientists are consulting with Weybridge 

scientists to determine the cause of the conflicting 

IHC test results.

• Several factors could cause or contribute to the 

discrepancy as follows:

o This animal had a very low level of infectivity and

therefore the sensitivity of USDA’s routine IHC 

test might not have been sufficient to detect the 

disease.

o Weybridge experts indicate that deposits of 

abnormal prion in the brain tissue were not 

uniformly distributed and were present at low 

concentration, which means that even adjacent 

samples of brain tissue might not give identical 

results.

Factsheet

Veterinary Services June 2005

APHIS
USDA Protocol Review:

• USDA will develop a protocol to conduct dual 

confirmatory tests, the IHC and the Western blot, 

when the screening test, the Biorad, returns an 

inconclusive result.

• USDA and Weybridge scientists are in agreement 

that the Biorad test is a very effective and appropriate 

screening test.

• USDA scientists will consult with Weybridge scientists 

to assess the array of antibodies available for use in 

IHC confirmatory tests to determine the most 

appropriate for use in United States confirmatory 

tests.Those consultations will be repeated periodically.

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national origin, sex, religion, age, disability, political beliefs, sexual

orientation, or marital or family status. (Not all prohibited bases

apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille,

large print, audiotape, etc.) should contact USDA’s TARGET

Center at (202) 720–2600 (voice and TDD).

To file a complaint of discrimination, write USDA, Director, Office

of Civil Rights, Room 326–W, Whitten Building, 1400

Independence Avenue, SW, Washington, DC 20250–9410 or call

(202) 720–5964 (voice and TDD). USDA is an equal opportunity

provider and employer.

United States Department of Agriculture • Animal and Plant Health Inspection Service • Safeguarding American Agriculture