Anti-Gliadin IgG ELISA Kit

Catalog No. ABIN1305150

Product Details

Target: Anti-Gliadin IgG

Reactivity: Giardia lamblia

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 1.0-100 U/mL

Minimum Detection Limit: 1 U/mL

Application: ELISA

Purpose: This microplate-based ELISA (enzyme linked immunosorbent assay) kit is intended for the qualitative detection of anti-Giardia lamblia IgG antibody in test sample. The assay is a useful tool in the aid of determination of Giardia lamblia infection in acute or chronic gastroenteritis. It is for professional use only.

Brand: ED™

Sample Type: Serum, Plasma

Analytical Method: Qualitative

Specificity: This assay does not detect human Anti-Giardia IgA or IgM, as well as other autoantibodies.

Components: 1. Giardia Antigen Coated Microplate
One bottle contains 30 mL of 3- fold concentrate. Before use the contents must be diluted with 870 mL of distilled water and mixed well. Upon dilution this yields a working wash solution containing a surfactant in phosphate-buffered saline with a non-azide preservative. The diluted wash buffer should be stored at room temperature and is stable until the expiration date on the kit box.

Material not included: 1. Precision single channel pipettes capable of delivering 10 µL, 50 µL, 100 µL, and 1000 µL, etc.
2. Repeating dispenser suitable for delivering 100 µL.
3. Disposable pipette tips suitable for above volume dispensing.
4. Disposable 12 x 75 mm or 13 x 100 glass or plastic tubes.
5. Disposable plastic 1000 mL bottle with cap.
6. Aluminum foil.
7. Deionized or distilled water.
8. Plastic microtiter well cover or polyethylene film.
9. ELISA multichannel wash bottle or automatic (semi-automatic) washing system.
10. Spectrophotometric microplate reader capable of reading absorbance at 450 nm.

Application Details

Sample Volume: 10 μL

Assay Time: 4 h

Plate: Pre-coated

Protocol: This ELISA is designed, developed and produced for the quantitative measurement of human anti-Giardia lamblia IgG in test specimen. The assay utilizes the microplate based enzyme immunoassay technique by coating highly purified and inactive Giardia lamblia antigen onto the wall of microtiter well. Assay standards, controls and unknown specimen are added to microtiter wells of microplate that was coated with a highly purified Giardia lamblia antigen on its wall. The Giardia lamblia antigen will be bound to the antibody in the liquid standards, controls and test samples. The unbound matrices are washed away and a HRP-conjugated antibody which specifically recognizes the specific subtype of human antibody (IgG) is added for further immunoreactions. After an incubation period, an immunocomplex of Giardia lamblia Antigen human Anti-Giardia IgG HRP-conjugated Anti-hIgG Antibody is formed if the human anti-Giardia IgG is present in the test sample. The unbound tracer antibody and other protein or buffer matrix are removed in the subsequent washing step. HRP-conjugated tracer antibody bound to the well is then incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the tracer antibody bound to the wall of each microtiter well is directly proportional to the amount of human Anti-Giardia lamblia IgG level in each test specimen.

Reagent Preparation:

Reagent Preparation (1) Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged.
(2) Concentrated Assay Buffer Concentrate must be diluted to working solution prior use. Please see REAGENTS section for details.
(3) ELISA Wash Concentrate must be diluted to working solution prior use. Please see REAGENTS section for details.

Sample Collection: Only 10 µL of human serum (or plasma) is required for Human Anti-giardia IgG measurement. No special preparation of individual is necessary prior to specimen collection. Whole blood should be collected and must be allowed to clot for minimum 30 minutes at room temperature before the serum is separated by centrifugation (850 ? 1500xg for 10 minutes). The serum should be separated from the clot within three hours of blood collection and transferred to a clean test tube. Serum samples should be stored at 2 ? 8 C up to 48 hours and at -20 °C or below for long term storage until measurement.

Sample Preparation:

Patient samples need to be diluted 1:100 with1x Assay Buffer before being measured.
(1) Label a test tube (12x75 mm) or a 1.5 mLplastic vial.
(2) Add 1 mL of assay buffer (1x) to each tube or vial.
(3) Add 10 μL of serum or plasma sample to the above tube.Note: If the test procedure is performed on an automated ELISA system, the supernatant must be particle-free by centrifuging the sample.

Assay Procedure:

(1) Place a sufficient number of Giardia antigen coated microwell strips in a frame.
(2) Test Configuration
(3) Add 100 µL of standards , controls and diluted patient serum samples into the designated microwell.
(4) Cover the plate with one plate sealer.
(5) Incubate plate at room temperature for 1 hour.
(6) Prepare working anti-hIgG 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 IgG Tracer Antibody in a clean test tube.
(7) 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.
(8) Add 100 µL of above diluted tracer antibody working solution to each of the wells.
(9) Cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light.
(10) Incubate plate at room temperature for 30 minutes.
(11) 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.
(12) Add 100 µL of ELISA HRP Substrate into each of the wells. (13) Cover the plate with a new plate sealer and also with aluminum foil to avoid exposure to light. (14) Incubate plate at room temperature for 15 minutes (15) Remove the aluminum foil and plate sealer. Add 100 µL of ELISA Stop Solution into each of the wells. Mix gently. (16) Read the absorbance at 450 nm within 10 minutes in a microplate reader

Calculation of Results:

1. Calculate the average absorbance for each pair of duplicate test results.
   2. Subtract the average absorbance of the calibrator 1 (0 U/mL) from the average absorbance of all other readings to obtain corrected absorbance.
   3. The calibrator curve is generated by the corrected absorbance of all calibrator levels on the ordinate against the calibrator concentration on the abscissa using point-to-point or log-log paper. Appropriate computer assisted data reduction programs may also be used for the calculation of results. The Giardia IgG concentrations for the controls and samples are read directly from the calibrator curve using their respective corrected absorbance. If log-log graphic paper or computer assisted data reduction program utilizing logarithmic transformation are used, sample having corrected absorbance between the 3.1 U/mL calibrator and the next highest calibrator should be calculated by the formula: Corrected absorbance (unknown) Value of unknown = x Value of the 2nd STD Corrected Absorbance

Assay Precision: The intra-assay precision is validated by measuring two samples in a single assay with 12-replicate determinations. The inter-assay precision is validated by measuring two samples in duplicate in 12 individual assays.

Restrictions: For Research Use only

Handling

Precaution of Use: The reagents must be used in a laboratory and is for professional use only. The source of material for reagents containing bovine serum albumin was derived in the contiguous 48 United States. It was obtained only from healthy donor animals maintained under veterinary supervision and found free of contagious diseases. Wear gloves while performing this assay and handle these reagents as if they are potentially infectious. Avoid contact with reagents containing TMB, hydrogen peroxide, or sulfuric acid. TMB may cause irritation to skin and mucous membranes and cause an allergic skin reaction. TMB is a suspected carcinogen. Sulfuric acid may cause severe irritation on contact with skin. Do not get in eyes, on skin, or on clothing. Do not ingest or inhale fumes. On contact, flush with copious amounts of water for at least 15 minutes. Use Good Laboratory Practices.

Storage: 4 °C

Target Details

Target: Anti-Gliadin IgG

Target Type: Antibody

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 cycts. 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 (IgA and IgG) against Giardia lamblia. Determination of human anti-giardia antibody may contribute to the aid of clinical diagnosis and understanding of the status of immune response for each individual.