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Understanding bioassays

cartoonBioassays are desired and can be useful tests that assist veterinarians in diagnosing disease and forming a treatment plans.  Generally, multiple tests over time are best and indicate if there is a treatment response or lack of effective therapy.

It is extremely important to understand what is being tested in an assay, how values are reported, and what the value means. This basic understanding gives veterinarians clues on how the assay will change over time in a tested animal.  A common question we get is “Why did the SAG titer (go up, not change)?” on a retested sample. This blog reviews the basics and after reading it you should be able to answer that question.  It is also good to stretch the envelope and understand new tools and what they can reveal about diseases in horses. Understanding new tools requires some background information, that is usually in accessible documents for those which are interested.

Bioassays differ in format and formats are as different as apples and oranges.  For example, enzyme linked immunosorbent assays, ELSIA’s, are different test systems (format) than immunofluorescent assays, IFAT’s.  Unless someone has published data comparing tests in a “head-to-head” experiment, direct comparisons between tests are not valid and this includes ELISA and IFAT testing. ELISA tests are different because they are designed to select different molecules. If you have a tool that detects apples, you won’t count oranges.  Likewise, if your tool detects oranges, you will miss the apples.  And if your experience and understanding is limited to one tool, without some investigation, you will miss what is possible. New tools can differentiate a Granny Smith from a McIntosh!

You may need a few definitions here for clarity.

Disease causing organisms elicit immune responses in infected animals, this response is called an adaptive response.  Adaptive responses can be an antibody or more complicated T-cell responses. We leave out the T-cell responses in this discussion even though they are important in polyneuritis equi pathology.  Antibodies are adaptive because they adapt the immune system to respond to a specific organism. There are also immune defenses, innate immune molecules called cytokines, that are released to fight off  infections before the body has identified the specific organism.  Cytokines are not specific, as opposed to adaptive immunity. A test measures molecules called analytes.  In antibody detecting tests, the protein that elicited the adaptive immune response is called an antigen.  An analyte can be an antibody or a cytokine or vitamin or toxic substance (lead is a good example). An analyte can be air.

Antibodies are very specific for the proteins that induced the response.  Organisms can share antigens, organisms as different as humans and mice can share antigens.  Antibodies to these common proteins don’t clearly differentiate organisms to genus or perhaps species.  Some antigens can be so specific they are used to identify a strain belonging to a species! Tests that differentiate organisms by an antibody response is called serotyping. A good example is S. neurona. There are three specific and dominant Surface AntiGens (SAG’s) of S. neurona, that define the genotype.  Because each SAG is genetically mutually exclusive, each protozoa only displays one of the three possible antigens on its surface.  When S. neurona infects the animal, an antibody is produced and directed against that SAG, the cause of the infection can be serotyped.

Keep in the back of your mind that opossums usually are infected with all three genotypes of S. neurona and horses generally are exposed to more than one genotype. S. neurona also elicits responses to numerous common antigens. Detecting responses to common antigens won’t distinguish between genus or species.  For example, on some testing formats shared antigens between S. neurona and S. fayeri and S. falcatula are detected and a positive result on the test won’t distinguish organisms causing EPM (S. neurona), EMS (S. fayeri) or no horse-disease at all (S. falcatula). Educated guesses are made based on dilutions and probability statistics.  Direct measures are preferred.

Other than antibody molecules, other analytes are measured in body fluids.  Analytes can be cytokines such as IL6, C reactive protein (CRP), vitamins, and neurofilament light (NfL).

Let’s get back to assay format. A Western Blot (WB) is a method to separate all the antigens from an organism by molecular size. The analyte (an antibody in the blood or CSF) reacts with the antigens and a pattern is detected.  The intensity of the reaction and experience with a pattern are factors on deciding a test positive status.  A confounding factor in WB’s are the size of some antigens.  It is no surprise that organisms have lots of similar sized proteins.  It isn’t a surprise that some types of separation gels will inadequately separate proteins that are similar in size, the conditions used to make the antigen preparation and even the conditions to run the assay have large impacts.   My PhD thesis demonstrated that SAG 1 ran on a WB with other similar sized proteins and using detergent in the assay decreased the amount of SAG 1 on the gel.  Not a new concept yet ignored in commercial testing, ultimately clouding the interpretation of SAG 1 infections. It shouldn’t be a big surprise that using S. neurona as the antigen source in a WB will give different results than using S. falcatula for  antigens.

The IFAT format uses a  S. neuronaSAG1 strain S2 in the test. To detect a SAG 5 or SAG 6 strain of S. neurona using the S. neuronaSAG1 strain S2 test, the interpretation must be based on common antigens.  That means S. fayeri antibodies found in horses will be positive on S. neuronaSAG1 strain S2 tests. There are several papers from different authors that discuss the data from serum/CSF analysis.  A statistical probability analysis can sometimes be used with test interpretation.  Be sure and know how positive values were obtained to validate the test, one or 100 or 1000 horses.

ELISA plateThe 2, 4/3 ELISA uses a chimeric (synthetically made antigen) and is a completely different assay than the SAG 1, SAG 5 or SAG 6 ELISA’s. The SAG 1, 5, 6 ELISA  uses recombinant proteins, folded in the native S. neurona proteins conformation (no detergent). The 2, 4/3 ELISA isn’t capable of serotyping S. neurona.  The 2, 4/3 protein will detect the SAG 2, SAG 3, and SAG 4 protein of S. fayeri.  The antigen we use to detect S. fayeri isn’t a surface antigen.  The S. fayeri assay we use employs the toxin from toxin producing strains of S. fayeri that may cause disease. For the skeptics, there are four papers available that discuss this toxin and its relationship to equine disease. ELISA tests can be specific enough to detect the infection stage of an the organism, such as Borrelia (Lyme disease).  The antigen displayed during acute infection is most interesting to veterinarians considering Lyme disease in a horse. Antibody detecting tests detect antibody against myelin protein 2 (MP2).  There are copious papers on this topic in people and horses, the protein, and disease associated with the protein.  The importance in running the test is the source and  native folding of MP2.

Now you know the keys to understanding bioassays are  format of the tests and antibody specificity used in each test. What else is important?

When will antibody levels change? It is expected that antibodies will go down depending on the immune status of the animal (naïve, first infection) or experienced (more than one infection) as well as exposure. It is known that antibodies against S. neurona in a naïve animal will go down in a couple of months.  The longevity of maternal antibodies in a foal are known. The SAG antibody levels in an experienced horse can take 10 months to recede, unless the animal is chronically exposed to the organism, an then the antibody levels will remain elevated. This information gives you the ability to use judicious testing and why, if tested too soon, an animal will remain positive or the antibody levels will go up.

Anti-myelin protein antibody levels can take 5 months to return to normal after the myelin isn’t presented to antibody producing cells. Levels of CRP and NfL will respond to the infection status, NfL responds to effective therapy and goes down in 7-10 days, CRP takes longer. The take home message is that antibody levels take a long time to go down while some of the other analytes decrease in a short time.  Also, understand that antibody levels are a measure of immune response and it is desirable to have protective antibodies.  It is undesirable to have autoimmune antibodies (anti-myelin protein antibodies). One needs to understand the disease pathology to effectively select and interpret tests.  Preventing protective immunity is not a good tactic while treating disease is a good strategy.

Putting it together.  Knowing what the test is designed to tell you and how those values are expected to change over time are fundamental.  It can not be said enough that there are no EPM tests.  None.  The tests that detect antibody against S. neurona do just that. They measure an antibody response to S. neurona but do not tell you the location in the body.  We used clinical disease, experimental infections, and recombinant antigens given as a vaccine to validate our SAG 1, SAG 5, and SAG 6 testing.  We report antibody levels (as a last dilution positive) against S. neurona when we run the SAG ELISAS.  We report a level of anti-toxin in serum or CSF in the S. fayeri ELISA test result.  The level of antibody against MP2 (or the reactive IL6 receptor, MPP protein) is an indication of demyelination.  An animal shouldn’t have these antibodies in their blood or CSF.  The MP2/MPP antibody is a measure of disease and the disease pathology.  Reporting a direct measure (mg/ml or micrograms/ml) for CRP and NfL are informative. CRP is a non-specific, acute phase cytokine response to inflammation and NfL is a measure of the axon damage in demyelinating and non-demyelinating polyneuropathies.