Adenovirus Antigen ELISA Kit

Catalog No. ABIN1305155

Product Details Adenovirus Antigen ELISA Kit

Target: Adenovirus Antigen (AdV Ag)

Reactivity: Adenovirus

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: This microplate-based ELISA (enzyme linked immunosorbent assay) kit is intended for the qualitative detection of Adenovirus antigen in feces. The assay is a useful tool in the diagnosis of active Adenovirus infection in acute or chronic gastroenteritis.

Brand: ED™

Sample Type: Fecal

Analytical Method: Qualitative

Specificity: This assay does not cross react to the following organisms: Rotavirus, Giardia iamblia, and Cryptosporordium parvum.

Characteristics: Gastrointestinal Disease

Components: 1. Anti-Adenovirus Antibody Coated Microplate. One bottle contains 30 mL of 10-fold concentrated buffer matrix with protein stabilizers and preservative. This reagent should be stored at 2-8 °C and is stable until the expiration date on the kit box. Before use the concentrated buffer must be diluted with 290 mL of distilled water and mixed well. Upon dilution this yields a working patient sample diluent containing a surfactant in phosphate buffered saline with a non-azide preservative. The diluted sample diluent can be stored at room temperature and is stable for 8 weeks. It can also be stored at 2-8 °C 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. 25 - 50 μL inoculating loop.3. Repeating dispenser suitable for delivering 100 μL.4. Disposable pipette tips suitable for above volume dispensing.5. Disposable 12 x 75 mm or 13 x 100 glass or plastic tubes.6. Disposable plastic 1000 mL bottle with caps
7. Aluminum foil
8. Deionized or distilled water
9. Plastic microtiter well cover or polyethylene film
10. ELISA multichannel wash bottle or automatic (semi-automatic) washing system.11. Spectrophotometric microplate reader capable of reading absorbance at 450 nm.

Application Details

Sample Volume: 0.1 mL

Assay Time: 4 h

Plate: Pre-coated

Protocol: This ELISA is designed, developed and produced for the qualitative measurement of Adenovirus antigen in test specimen. The assay utilizes the microplate-based enzyme immunoassay technique by coating highly purified antibody onto the wall of microtiter well. Assay controls and fecal specimen, as well as HRP-conjugated monoclonal antibody that specifically recognizes the inner capsid protein of the Adenoviruses are added to microtiter wells of microplate that was coated with a highly purified polyclonal anti-Adenovirus antibody on its wall. After an incubation period an immunocomplex of Anti-Adenovirus Antibody Adenovirus Antigen HRP-conjugated Anti-Adenovirus Tracer Antibody is formed if there is Adenovirus antigen 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 Adenovirus captured on the wall of each microtiter well is directly proportional to the amount of Adenovirus antigen level in each test specimen.

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) ELISA Wash Concentrate must be diluted to working solution prior use. Please see REAGENTS section for details.
(3) Concentrated Patient Sample Diluent must be diluted to working solution prior use. Please see REAGENTS section for details.

Sample Collection: 1. Stool specimens can be collected at any time of the day.2. Collect a random sample of feces into a fecal sample collection container or tube or cup with an aid of a clean, dry cup or plastic spoon or toilet paper.3. It is required to collect minimum 0.1 mL liquid stool sample or 0.1 g solid sample.4. The specimen is ready for testing, transportation or storage. It can be stored at 2-8 °C for up to 3 days and at frozen condition (-20 °C) for longer storage.

Sample Preparation:

(1) Label a test tube (12x75 mm) or a 1.5 mLplastic vial.
(2) Add 1 mL of the diluted Patient Sample Diluent to each tube or vial.
(3) Add 100 μL of liquid stool sample to the above tube.(4) With solid stool sample, take an equivalent amount (about 50 100 mg) with a spatula or a disposable inoculation loop. Suspend the solid stool sample with 1 mL patient sample diluent and mix well in a vortex mixer. Allow the diluted sample to sediment for about 5 minutes. The supernatant can be directly used in the assay.(5) If the test procedure is performed on an automated ELISA system, the supernatant must be particle-free by centrifuging the sample at 5000 rpm (2000 2500 g) for 5 minutes.

Assay Procedure:

(1) Place a sufficient number of anti-Adenovirus antibody coated microwell strips in a holder to run Adenovirus controls and unknown samples in duplicate.
(2) Test Configuration
(3) Prepare working anti-Adenovirus tracer antibody working solution by 1:21 fold dilution of the Anti-Adenovirus Tracer Antibody (30186) with the Tracer Antibody Diluent. For each strip, it is required to mix 0.5 mL of Tracer Antibody Diluent with 25 μL of Tracer Antibody in a clean test tube.
(4) Add 100 μL of controls and diluted patient stool samples into each designated microwell.
(5) Add 50 μL of above diluted tracer antibody working solution to each of the wells.
(6) Cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light.
(7) Incubate plate at room temperature for 1 hour.
(8) 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.
(9) Add 100 μL of ELISA HRP Substrate into each of the wells.
(10) Cover the plate with aluminum foil to avoid exposure to light.
(11) Incubate plate at room temperature for 10 to 20 minutes.
(12) Remove the aluminum foil. Add 100 μL of ELISA Stop Solution into each of the wells. Mix gently.
(13) 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. Calculate the cut-off:The positive cut-off and the negative cut-off is established by using following formula.Positive Cut-Off = 1.1 x (mean extinction of negative control + 0.08)Negative Cut-Off = 0.9 x (mean extinction of negative control + 0.06)3. Interpret test result? Positive: patient sample extinction is greater than the Positive Cut-Off.? Negative: patient sample extinction is less than the Negative Cut-Off.? Equivocal: patient sample extinction is between the Positive Cut-Off and the Negative Cut-Off.

Assay Precision: The reproducibility of this assay was validated by measuring two positive samples and one negative sample in 5 differenct assays run on different days. The results showed a consistent result interpretation for all the samples.

Restrictions: For Research Use only

Handling

Precaution of Use: The reagents must be used in laboratory and are for professional use only. The source material for reagents containing bovine serum 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 for Adenovirus Antigen

Target: Adenovirus Antigen (AdV Ag)

Alternative Name: Adenovirus Antigen (AdV Ag Products)

Target Type: Viral Protein

Background: Acute diarrheal disease in young children is a major cause of morbidity worldwide and is a leading cause of mortality in developing countries. Research has shown that enteric adenoviruses, primarily Ad40 and Ad41, are a leading cause of diarrhea in many of these children, second only to the rotaviruses. However many different symptoms can manifest, depending on the type of infecting Adenovirus. There are 49 distinct serotypes that can cause infections in humans. The diarrhea resulting from enteric adenoviruses is longer in duration than that caused by the rotaviruses, usually lasting 7 - 8 days. Adenovirus infections often show up as conjunctivitis, tonsillitis (which may look exactly like strep throat and cannot be distinguished from strep except by throat culture), an ear infection, or croup. Adenoviruses can also cause gastroenteritis (stomach flu). A combination of conjunctivitis and tonsillitis is particularly common with adenovirus infections. Small children are especially prone to develop adenovirus bronchiolitis or pneumonia, both of which can be severe. In babies, adenoviruses can also cause coughing fits that are almost exactly like whooping cough. Adenoviruses can also lead to viral meningitis or encephalitis. Rarely, adenovirus causes inflammation of the urinary bladder (also known as cystitis), producing blood in the urine. In children, adenoviruses may cause acute upper respiratory infections with fever and runny nose. Adenovirus types 1, 2, 3, 5, and 6 are responsible for most of these infections.Specific diagnosis of the Adenovirus infection is made by identification of the virus in the patient's stool. Enzyme linked immunsorbent assay (ELISA) is the test most widely used to screen clinical specimens. Electron microscopy and polyacrylamide gel electrophoresis are used in some laboratories in addition or as an alternative to ELISA.