# CAE UPDATE-- ELISA VS. AGID Testing



## sweetgoatmama (Dec 10, 2008)

CAE comments from WSU Updated 2008

Posted with premission of the author -
John VanderSchalie
Associate in Immunodiagnostics
Serology Laboratory Manager
Washington Animal Disease Diagnostic Laboratory
Pullman, Washington

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I will try to address a couple of the misconceptions regarding CAE testing. I must qualify my statements below by saying that I am not a veterinarian, but a microbiologist who has been in the serology field for over 30 years. I developed a first generation ELISA test for CAE, and for the last five or so years we have been using a second generation ELISA test, which I'll explain
below.
The small ruminant lentiviruses, of which CAE is a member, are somewhat unique. Of all the animals infected, only 10-15% of them will ever show the primary sign of the disease (arthritis). In fact, the goats that get arthritis, are the ones that mount a specific type of immune response to the virus, and their immune response is the cause the disease. This is very unlike other
viruses, in which the immune response cures the disease and gives immunity to future infections. Once infected, the animal is infected for life, and has a great chance of passing the infection to offspring, primarily through the colostrum.
There is also a misconception about testing animals when pregnant. It is generally regarded that about 20-25% of serum immunoglobulins (antibodies) are taken from the circulating blood stream and packed into the colostrum late in pregnancy. If an animal was very recently infected with a lentivirus, there is the possibility that a weak positive testing goat could drop into the negative zone. However, if the animal was infected for more than 4-6 weeks, the amount of antibody to the primary antigen (gp-135, see below) is so high that it will not drop the levels out of the positive zone on this test. And, in fact, often towards the end of pregnancy, the animal makes up for this antibody packing into the colostrum by making more antibodies than it would if not
pregnant.
Now, a word on how the current competitive ELISA that we use works. The predominant antigen that the CAE virus makes and releases is the gp-135 antigen. Goats typically respond with 100 fold more antibody to the gp-135 antigen than to any other of the CAE viral antigens. The "gp" stands for glyco-protein, and it's weight is 135,000 kilo-daltons. To make the test kits,
this gp-135 antigen is attached to the bottom of 96 well plates. The goat serum being tested is put into this well, and if the goat has antibodies to the gp-135 antigen, they will specifically attach. The key part which makes a "competitive" ELISA so specific comes next. A monoclonal antibody, obtained from lymphocyte clones which only recognize one *very specific* region on that
gp-135 antigen is put in the test and control wells. Going back over the procedure, after the serum has been in the gp! -135 coated well for an hour, the well is washed a number of times to remove any unattached goat antibody, and this monoclonal antibody with enzyme attached is added to the well for 30
minutes. It can only attach itself to that highly specific site on the gp-135 antigen if that site is not covered up by the test-goat's antibodies. It must "compete" for that site, hence the term, competitive ELISA. This is where the high specificity is obtained. If the monoclonal attaches, it means that the goat serum being tested did NOT have any antibody to that specific site, because
it did not block the monoclonal antibodies from attaching. The c-ELISA is considered the most sensitive and specific assay available world-wide for testing goats for CAE. Through testing large numbers of known positive and known negative goats, a positive/negative cut-off number was selected. At the
cut off number, the sensitivity is 100% and specificity has been calculated to be 99.6%. That says that there are predicted to be four false positives for every 1,000 tested, but all the true positives will be identified. The cut point is selected to have the highest sensitivity and specificity. If one is trying to eradicate a virus, the test should be skewed to a higher sensitivity. 
Is it more dangerous to have a false negative (infected animal), than a false positive (uninfected animal) in your herd? The competitive ELISA is by far the best test available in terms of sensitivity and specificity. The AGID test has a very high specificity (positives are truly positive), but a far lower sensitivity. Infected animals will be missed because they do not have high enough antibody levels to be detected with that test. In a scientific paper by USDA scientists in the 1980's, the sensitivity of the AGID test is only 56%! I believe the current AGID tests are better than this, but no where near approaching the sensitivity and specificity of the competit! ive ELISA test. 
The competitive ELISA WADDL uses is VALIDATED AND LICENSED BY USDA, with sensitivity and specificity listed, whereas the PCR-based assays are not. 
Please check into the sensitivity and specificity of the other tests available for this disease.
Many people have eradicated CAE from their herds by good management and serum testing. It is not an easy task, but can be done. We recommend that animals testing positive be segregated and re-tested before management decisions are made. Animals newly introduced into a herd should be segregated and have
two negative tests a number of weeks apart before introduction into the herd.
The lab is always ready to consult on CAE questions. Please have
concerned individuals contact either Dr. Evermann or myself with questions. CAE eradication is dependent on a reliable assay (which we have) and owner compliance.

John VanderSchalie
Associate in Immunodiagnostics
Serology Laboratory Manager
Washington Animal Disease Diagnostic Laboratory
Pullman, Washington
509 335-3429
[email protected]


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