<<Disclaimer: Verify this information before applying it to your situation.>>

Two methods used to detect antibodies are ELISA and IFA.

ELISA = enzyme linked immunosorbent assay

ELISA can be used to detect antigen (gluten) or antibody. For 
diagnosis we look for antibody. Antigen is bound to a surface. Sample 
that may or may not contain antibodies to the antigen (ie. your blood 
sample) is washed over the surface. If antibody is there it will bind 
to the antigen on the surface. Another antibody that binds to the 
first antibody is added (an anti-antibody), but this one has a special 
enzyme attached to it. After washing the plate, only blood samples 
that contained the antibodies of interest will have the special enzyme 
attached to the surface. Now a specific chemical can be added that 
interacts with the enzyme to produce a detectable signal (signal is 
accurately “counted” with an instrument).

IFA = immunofluorescence assay
  
Some antibodies can be chemically "tagged" with a fluorophore (a very 
pretty dye). So compared to the above ELISA technique there is no need 
to add a chemical to a surface-bound enzyme in order to produce a 
detectable signal. Fewer steps = better, plus you can see positive 
results with the naked eye and fluorophores are just fun to work with! 
Fluorescence is accurately “counted” with an instrument.

Dilutions and titers.

Serial dilutions are used to standardize the test results. If everyone 
reported a number that some instrument spit out it would be difficult 
to compare results from different instruments (each of which might be 
detecting a unique signal depending on the method employed). The 
number that is reported looks something like 1:X, where X is some 
number referred to as the titer.

The standard two-fold dilution works like this: sample tubes are 
prepared so that each contains half the quantity of antibody (your 
serum sample) that is found in the previous tube. So the second tube 
contains half the amount of antibody, the third tube contains a 
quarter of the amount of antibody, the fourth tube contains an eighth, 
the fifth contains a sixteenth, the sixth a thirty-second, the seventh 
tube a sixty-fourth, etc… Each tube contains less and less antibody so 
signal should be proportionately weaker and weaker. The point at which 
no signal is detected is referred to as the endpoint. “Positive” or 
“negative” is specific to a particular antibody and method (not the 
same for AGA as EMA). Let’s say a positive result for kickme disease 
is reportedly 1:16 or greater. If Mary’s titer is 8 and Joe’s is 64 it 
means that Joe has kickme disease and Mary does not. If MaryJo's 
result was 1:128 then her case of kickme might be considered more 
severe than Joe's.

For EMA tests a two-fold dilution is used starting at 1:5 and going to 
1:40. A titer equal to or greater than 40 is positive, with “weak 
positives” at 5, 10, and 20.

Cheers,

Megan

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