Anti-Giardia Iamblia IgM ELISA

Additional Information

Assay Background

Giardia lamblia (also known as Giardia intestinalis) has a characteristic tear-drop shape and measures 10-15 µm in length. It has twin nuclei and an adhesive disk which is a rigid structure reinforced by supelicular microtubules. There are two median bodies of unknown function, but their shape is important for differentiating between species. There are 4 pairs of flagella, one anterior pair, two posterior pairs and a caudal pair. These organisms have no mitochondria, endoplasmic reticulum, golgi, or lysosomes. Giardia has a two-stage life cycle consisting of trophozoite and cyst. The life cycle begins with ingested cysts, which release trophozoites (10-20 µm x 5-15 µm) in the duodenum. These trophozoites attach to the surface of the intestinal epithelium using a ventral sucking disk and then reproduce by binary fission. The trigger for encystment is unclear, but the process results in the inactive, environmentally resistant form of Giardia — a cyst (11-14 µm x 7-10 µm) that is excreted in feces.

Giardiasis is a diarrheal illness caused by Giardia lamblia, after ingestion of Giardia cysts. Once a person has been infected with Giardia, the parasite lives in the intestine and is passed in the stool. Millions of germs can be released in a bowel movement from an infected human or animal. Giardia is found in soil, food, water, or surfaces that have been contaminated with the feces from infected humans or animals. Because the parasite is protected by an outer shell, it can survive outside the body and in the environment for long periods of time. Because it is spread world-wide, Giardia lamblia has become one of the most important causes of chronic diarrheas. About 15-20% of children under age ten years and 19% of male homosexuals have been infected. Giardia infection can cause a variety of intestinal symptoms either acute or chronic, which include diarrhea, gas or flatulence, greasy stools that tend to float, stomach cramps, upset stomach or nausea. These symptoms may lead to weight loss and dehydration. Some people with giardiasis have no symptoms at all. Those asymptomatic cases still shed Giardia cysts. Generally, symptoms of giardiasis begin 1 to 2 weeks after becoming infected and they may last 2 to 6 weeks.

Despite the fact that Giardia is essentially a luminal pathogen in the gut it does evoke both systemic and local immune responses. Current between serum and secretory antibody responses remains unclear, the presence of anti-Giardia antibodies in serum would be in any way indicative of the development of protective immunity. Evidence emphasizes the importance of secretory antibody for clearance of the pathogen, although other cell-mediated effector mechanisms are also likely to be involved.

Recent studies have found that about 86% of infected patients develop serum antibodies against Giardia lamblia.

Assay Procedure

1. Place a sufficient number of Giardia antigen coated microwell strips  in a frame.
2. Add 100 µL of standards, controls and diluted patient serum samples into the designated microwell.
3. Cover the plate with one plate sealer.
4. Incubate plate at room temperature for 1 hour.
5. Prepare working anti-hIgM Tracer Antibody Working Solution by 1:21 fold dilution of the tracer antibody with the Tracer Antibody Diluent For each strip, it is required to mix 1 mL of Tracer Antibody Diluent with 50 µL of IgM Tracer Antibody in a clean test tube.
6. Remove the plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL to 400 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
7. Add 100 µL of above diluted tracer antibody working solution to each of the wells.
8. Cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light.
9. Incubate plate at room temperature for 30 minutes.
10. Remove the plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL to 400 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
11. Add 100 µL of ELISA HRP Substrate into each of the wells.
12. Cover the plate with a new plate sealer and also with aluminum foil to avoid exposure to light.
13. Incubate plate at room temperature for 15 minutes.
14. Remove the aluminum foil and plate sealer.  Add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
15. Read the absorbance at 450 nm within 10 minutes in a microplate reader.

Standard Curve