How It’s Done: The IgG Test

By Kristi Murdock

Since I ordered the IgG test supplies from Triple J Farms, I got to see how it all works, and I thought you might be interested.

To perform the test, one first draws blood from the llama into a regular, “red-top” collection tube.  Seven ml. is plenty.  (Hint — if you are performing the test on a new cria and plan to submit a DNA card to the International Lama Registry, this is a great time to get blood for that, as well!) 

The blood is allowed to clot in the tube at room temperature, and then spun briefly in a centrifuge to separate the clear serum from the red cells.  The serum is drawn off with a syringe.

This is what the test plate looks like:

It’s called a Radial Immunodiffusion, or RID Plate.  It contains a special gel that contains antigens, which react when combined with antibodies, the stuff that fights off infection.  There are 24 tiny holes in the cover of the plate, forming “wells” into the antigen. 

The test kit also comes with four vials of serum for which the IgG level has already been calculated.  They look like this:

You can use one or more of these to establish “benchmark” results on your test kit.  With my kit, we used the first four wells and “tested” each of the provided serums. 

To test a serum, one uses a tiny tube and plunger combination that you can also buy from Triple J Farms.  The container of 5 ul. Pipette tubes, a single tube and the plunger are shown below:

The plunger is inserted into the tube, and then the tip of the tube is put into the serum to be tested.  As the plunger is withdrawn, the serum rises into the tube.  There is a small mark on the tube to show you how much serum to draw.

The tip of the tube is then put into one of the wells on the RID plate, and then depressed, putting that tiny, precise amount of serum into the well.  The plate is then set aside.  Preliminary results can be read at 12 hours, and final results can be read after 24 hours.  What happens during this period is that a ring develops around the well, the result of the antibodies that are in the serum reacting with the antigens in the plate.  The bigger the ring, the more antibodies were in the llama blood.

To read the result, one uses a magnifying glass that has a scale printed on it, called a comparator.  The maginification used is 7x.  The comparator looks a lot like a small shot glass, but is quite a bit more expensive, and can also be purchased from Triple J Farms.

This is what the comparator looks like:

The comparator is held up to the RID plate, and I found it easiest to hold the whole thing up to a window, so that the plate is back-lit.

The resulting view is something like this:

The cross-hairs of the comparator are lined up with the edge of the precipitin ring on the plate, so that you can measure its diameter.

The test kit includes a printed table so that you can look up the measurement and see the IgG level for that size of ring on that specific plate (the level of antigen can vary by plate batch, so there is no “absolute” reference you can use across all plates). 

If you’re in a hurry, you can get a rough idea of the IgG level by comparing the ring to the results of the test vials, if you put them into the plate as we did.  However, I do recommend you measure the results of the test vials to make sure they correspond to the ring size specified in the table.  We didn’t fill the well correctly with test serum #2 and the resulting ring was smaller than it should have been.  However, we used the erroneous result to compare an actual test result against, and so thought that the cria’s blood tested higher in antibodies than it really did.

The plate can be stored in the refrigerator until it expires according to the date on the packaging.  I ordered mine in May, 2003 and it expired in July, 2004, so I had 24 tests to use any way I wanted during that period.

You can purchase plasma in either medium (2000-2499 mg/dl) or high (2500-3500 mg/dl) IgG concentrations.  The folks at Triple J Farms have come up with a formula that, based on the weight of your cria and the results of the IgG test, will tell you how much plasma of a particular IgG level must be transfused into the cria to raise the IgG level by a particular amount (see their website below).  For example, I used the formula to determine that it would take about 500 ml of 3400 mg/dl IgG plasma to bring my cria from her 170 mg/dl to a target 800 mg/dl.  When asked about the "shelf life" of frozen plasma, Don Jorgensen at Triple J said "We have used plasma frozen and not thawed for three years with no bad effects. We tell people to try to use it in 1 year. IgG which contains the protective antibodies most people desire is very stable and I have documented it's stability with preservative at 25 years."

The transfusion process itself isn’t nearly as bad as I’d thought — sticking a big needle into somebody’s gut sounds scary, but, in actually, it was much less intrusive and much quicker than giving IV fluids.  Triple J Farms also has guidelines to provide to your Vet for the transfusion, and they sell a filtered IV administration set and an “infusion tube” (a 12-gauge blunt end “needle” with two side ports, of the type used to administer medication into cows’ teats).  First, you make sure your plasma is thawed and warmed to body temperature.  The Vet shears a small area on the cria’s side, just ahead of the hind leg, cleans and disinfects the area thoroughly, numbs the area with a topical anesthetic, and makes a small incision with a scalpel.  The “needle” is used to punch into the intraperitoneal cavity, and the IV hookup is then turned on to drain the plasma into the cria.    We had a small pen set up, and the cria was able to move about in the pen with the Vet holding the plasma bag above her head during the infusion.  After the bag was empty, the needle was removed, and the cria rejoined her mother and started to nurse.  She got antibiotic shots for the next three days, just to help prevent any infection in the area.

For more information on obtaining all the items described, as well as directions on how to test IgG levels, how to calculate how much plasma to give to a llama to increase IgG levels to a particular level, and how to transfuse plasma, contact

kent@kentlabs.com

Triple J Farms

777 Jorgensen Place

Bellingham, WA  98226

360-398-9512

603-954-6333

http://www.kentlabs.com/triplej.html