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HBcAg Elisa test detect Anti-HBe Antibody,96wells/kit, Elisa Sandwich method, for quantitative measurement
|NAME AND INTENDED USE|
HBcAb ELISA Kit is an in vitro enzyme linked immunoassay supplied for the detection of HBcAb in human serum or plasma. It is intended for use in clinical laboratories for diagnosis and management of patients related to infection with hepatitis B virus.
Hepatitis B is an infectious illness caused by hepatitis B virus (HBV) which is an envelope, double-stranded DNA virus belonging to the Hepadnaviridae family and is recognized as the major cause of blood transmitted hepatitis together with hepatitis C virus (HCV). HBV is excreted in body fluids such as semen, saliva, blood and urine in persons with acute or chronic infection. The hepatitis B virus is known as a blood-borne virus because it is transmitted from one person to another via blood or fluids contaminated with blood. Transmission of hepatitis B virus results from exposure to infectious blood or body fluids.When HBV invades the body it causes liver damage through induction of auto-immunity. Three immunity has been found, surface antigen (HBsAg)/HBsAb, core antigen (HBcAg)/HBcAb and antigen (HBeAg)/HBeAb. As it is difficult to detect the core antigen in the serum, the other five are been done to diagnosing HBV. Hepatitis B “core” antigen (HBcAg) is a major component of the viral structure. Antibodies to HBcAg (anti-HBc total antibody, and IgM) appear shortly after the appearance of HBsAg and persist for life both in persons who have recovered from a hepatitis B infection and in those who develop HBsAg-carrier status but in rare cases, a HBV infection can also run its course without the appearance of immunologically detectable anti-HBc (usually in immunosuppressed patients). Anti-HBc is a marker of acute, chronic or resolved HBV infection and screening for anti-HBc provides with information on the prevalence of the disease in different groups. In the absence of other hepatitis B markers (HBsAg-negative persons), anti-HBc may be the only indication of an existing hepatitis B viral infection.
|PRINCIPLES OF THE ASSAY|
This HBcAb ELISA Kit is based on solid phase, one step incubation competitive principle method. Anti-HBc if present in the sample, compete with monoclonal anti-HBc conjugated to horseradish peroxidase (HRP-Conjugate) for a fixed amount of purified HBcAg pre-coated in the wells. When no anti-HBc presents in the sample, the HRP-conjugated anti-HBc will be bound with the antigens inside the wells and any unbound HRP-Conjugate is removed during washing.
The enzyme substrate, tetramethylbenzidine (TMB), is added upon incubation the substrate will be hydrolyzed by the bound enzyme and a blue or blue-green color develops in wells containing specific antibodies to HBcAg. The enzyme reaction is stopped by the addition of sulphuric acid. The intensity of color developed is read spectrophotometrically at 450nm (450 nm/630 nm) and is proportional to the amount of antibodies present in the specimen. No or low color developing suggests for presence of antibodies to HBcAg in the sample.
|MATERIALS REQUIRED BUT NOT PROVIDED|
1. Micropipettes: 0.02, 0.05, 0.10, 0.15, 0.20, and 1.0 ml.
2. Disposable pipette tips.
3. Distilled or deionized water.
4. Humidified Box capable of maintaining 37°C
5. Absorbent paper or paper towel.
6. Micro titer plate or strip-well washer
7. Micro titer plate reader with 450nm (or 450 nm/630 nm) wavelength
|SAMPLE COLLECTION AND PRESERVATION|
Blood serum samples are routinely prepared form vein. Blood plasma samples are routinely prepared with routine amount of anticoagulant such as heparin or sodium citrate. Sample can be stored at 4°C if tested within two days. Specimens not required within 3 days should be stored at -20°C. Avoid hemolysis and repetitive freeze and thaw of samples. Samples with cloud or precipitation should be centrifugated or filtered before test. Prevent serum from bacterium contamination during collecting and storing.
1. Bring ELISA Kit (all reagents), and Specimens to room temperature before use (approximately 30 minutes).
2. Check the Wash buffer concentrate for the presence of salt crystals. If crystals have formed in the solution, resolubilize by warming at 37°C until crystals dissolve. Dilute concentrated wash buffer 1:19 with ddH2O. Use only clean vessels to dilute the buffer.
3. For each test, set one blank, two positive and two negative controls. Add 50μl Positive control, Negative control, and Specimen into their respective wells. Add 50μl of HRP-Conjugate to each well except the Blank and mix by tapping the plate gently. Neither samples nor HRP-Conjugate should be added into the Blank well.
4. Cover wells with seal paper, and place the micro titer plate into a humidified box and incubate at 37°C for 30 min.
5. Discard the liquid in all wells and fill the wells with wash solution. Lay aside for 15 seconds, discard the liquid in all wells and fill the wells with wash solution. Repeat 5 times and blocking the rim of wells on absorbent paper for a few seconds wells after last wash.
6. Add 50μl substrate A and B respectively to each well including the blank well. Mix gently, protected from light and incubates 15 minutes at 37°C.
7. Add one drop (50ul) of Stop Solution to each well including blank well to stop the color reaction.
8. Read the OD value at 450 nm/630 nm with dual filter plate reader. It is option to read the OD value at 450 nm with single filter plate reader. (Using the OD value of the blank well to correct all the OD reading from all wells)
|ORIENT NEW LIFE MEDICAL CO., LTD.|
|Email:||Jerry @ newlifebiotest .com|
Contact Person: Jerry Meng