Intended Use
This microplate based ELISA (enzyme linked immunosorbent assay) kit is intended for the qualitative and quantitative detection of rotavirus antigen in feces. Specifically, this test detects VP6 of the viral capsid and is useful in detection of type A rotavirus infection. This kit is for research use only.
Contents of Kit
1. Anti-Rotavirus Antibody Coated Microplate
Microplate coated with a highly purified anti-rotavirus antibody.
Qty: 1 × 96 well microplate
Storage: 2-8°C
Preparation: Ready to Use.
2. Anti-Rotavirus Tracer Antibody
HRP-conjugated monoclonal anti-rotavirus tracer antibody in a stabilized protein matrix.
Qty: 1 × 12 mL
Storage: 2-8°C
Preparation: Ready to Use.
3. ELISA Wash Concentrate
Surfactant in a phosphate buffered saline with non-azide preservative.
Qty: 1 × 30 mL
Storage: 2-25°C
Preparation: 30× Concentrate. The contents must be diluted with 870 mL distilled water and mixed well before use.
4. ELISA HRP Substrate
Tetramethylbenzidine (TMB) with stabilized hydrogen peroxide
Qty: 1 × 12mL
Storage 2-8°C
Preparation: Ready to Use
5. ELISA Stop Solution: 0.5 M sulfuric acid
Qty: 1 × 12mL
Storage 2-25°C
Preparation Ready to Use
6. Rotavirus Antigen Calibrator Level 6: 1.5 mL of a calibrator in bovine serum albumin-based matrix with a non-azide preservative.
Qty: 1 × Vial
Storage: 2-8°C
Preparation: Ready to Use
7. Concentrated Patient Sample Diluent
Concentrated buffer matrix with protein stabilizers and preservative. Upon dilution this yields a working calibrator 6 matrix buffer, negative control, and 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.
Qty: 30 mL
Storage: 2-8°C
Preparation: 10× Concentrate. The contents must be diluted with 270 mL distilled water and mixed well before use.
Storage
This test kit must be stored at 2 -8°C upon receipt. For the expiration date of the kit refer to the label on the kit box. All components are stable until this expiration date.
General Description
Rotaviruses are the main and the most important pathogens that cause of non-bacterial acute gastroenteritis and diarrhea, especially in children from 6 months to 2 years of age, premature infants, the elderly, and the immunocompromised individuals. Rotaviruses have been identified in almost 40% of the faces of children with gastroenteritis. Rotavirus is the cause of up to 50% of the hospitalized cases of diarrhea in infant and young children. Almost every child has been infected with rotavirus by age 5. Over 3 million cases of rotavirus gastroenteritis occur annually in the US. There are about 120 million rotavirus infections every year worldwide and that causes the death of 600,000 to 650,000 children. Study also indicates that a high frequency of rotavirus infections may increase the risk of celiac disease autoimmunity in childhood in genetically predisposed individuals.
Rotaviruses have a genome consisting of 11 double-stranded RNA segments surrounded by a distinctive three-layered icosahedral protein capsid. The first layer is formed by the protein VP2, with each vertex having a copy of the proteins VP1 and VP3. The second layer is formed by the protein VP6. The outermost protein layer is composed of the structural glycoprotein VP7 and the spike protein VP4. Viral particles are up to 100 nm in diameter and have a buoyant density of 1.36 g/ml in CsCl. Rotaviruses tend to affect gastrointestinal epithelial cells that are at the tip of the villus. Their triple protein coats make them very resistant to the normally prohibitive pH of the stomach, and also digestive enzymes (lipases and proteases) in the gastrointestinal tract. During the infection, rotavirus produces mRNA to support both protein translation and genome replication.
Rotavirus is transmitted by oral-fecal contact with an incubation period of 1-3 days. Characteristic symptoms include vomiting, hydrodiarrhoea for between 3 and 8 days, high temperature and stomach pains. A large amount of rotavirus particles is shed during infection.
Specific diagnosis of the rotavirus 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.
Standard Curve
1. Quantitative MeasurementA typical absorbance data and the resulting calibration curve from Fecal rotavirus antigen ELISA are represented.
Note: This curve should not be used in lieu of calibration curve run with each assay.
2. Qualitative Measurement:
Positive Cut-Off = 1.1 × (0.074 + 0.08) = 0.1694Negative Cut-Off = 0.9 × (0.074 + 0.06) = 0.1206
Citations
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