Specifications

Enzyme-linked immunosorbent assay for the detection HBeAg in serum or plasma HBeAg EIA is a qualitative enzyme immunoassay for t

HBeAg ELISA Kit

 

Enzyme-linked immunosorbent assay for the detection HBeAg in serum or plasma

INTENDED USE

 

HBeAg EIA is a qualitative enzyme immunoassay for the detection of HBeAg in human serum or plasma.

 

SUMMARY AND PRINCIPLE OF THE TEST

 

The Hepatitis B e antigen is found to be related to Hepatitis B viral infection. HBeAg is usually found in the early phase of Hepatitis B viral infection. The titer of HBeAg rises rapidly during the period of virus replication. The presence of HBeAg correlates with increasing numbers of infectious viruses (Dane particles) and the presence of viral specific DNA polymerase in serum. During the HBeAg positive stage, hepatitis B patients are at increasing risk of transmitting the virus to their contacts. Persistent presence of HBeAg in the hepatitis B virus carrier is often associated with chronic active hepatitis.

 

The HBeAg EIA is a sandwich immunoassay, which employs monoclonal and polyclonal antibodies specific for HBeAg. Microtiter wells are coated with monoclonal antibodies specific for HBeAg. A serum specimen is added to the antibody coated microtiter wells together with enzyme conjugated polyclonal antibodies. HBeAg, if present, will form an antibody-HBeAg-antibody-enzyme complex. The plate is then washed to remove unbound materials. Finally, a solution of substrate is added to the wells and incubated. A blue color will develop in proportion to the amount of HBeAg present in the specimen. The enzyme substrate reaction can be stopped and the result is visualized by the naked eye or obtained by EIA plate reader for absorbance at wavelength of 450 nm (450 nm/630 nm).

 

REAGENTS

 

Materials provided with the kits:

1. Microtiter well coated with monoclonal anti-HBe: 96 tests in one bag.
2. Negative Control: 0.5ml HBeAg Negative Control.
3. Positive Control: 0.5ml HBeAg Positive Control.
4. Enzyme Conjugate:.6 ml containing HRP-conjugated-anti-HBe
5. Wash Buffer Concentrate (20 x): 20ml for 96 tests. The buffer should be diluted 20 times with distilled water before use.
6. Substrate Solution A: 6 ml HRP Substrate for 96 tests.
7. Substrate Solution B: 6 ml TMB Chromagen Substrate for 96 tests.
8. Stop Solution: One bottle of 6ml 2N Sulfuric Acid

 

Materials required but not provided:

1. Precision pipettes: 0.02, 0.05, 0.10, 0.15, 0.20, and 1.0 ml.
2. Disposable pipette tips.
3. Distilled water.
4. HumidifiedBox capable of maintaining 37°C
5. Absorbent paper or paper towel.
6. Micro titer plate or strip-well washer
7. Micro titer plate reader.

 

PRECAUTION FOR USERS

 

1. For in-vitro diagnostic use only.
2. Must not use kit beyond the expiration date.
3. Do not mix components from kits with different lot number.
4. Avoid microbial contamination of reagents.
5. Do not pipet reagent by mouth and no smoking or eating while performing assays.
6. Wear gloves during the whole process and avoid reagents or specimen spilling-out.
7. Wipe up the spills using 5% hypochlorite solution.
8. Decontaminate all liquids or solid wastes before deposing.

    

   SPECIMEN COLLECTION AND PREPARATION

    

   Serum should be prepared from a whole blood specimen obtained by acceptable medical techniques. Either serum or plasma can be used in this test. Remove serum or plasma from the clot or blood cells as soon as possible to avoid hemolysis. Specimen with extensive particulate should be clarified by centrifugation prior to use. Specimen frozen at -20°C or colder may be used. Avoid repeated freeze thaw.

    

   STORAGE OF TEST KIT

    

   Unopened test kits should be stored at 2-8C upon receipt and the microtiter plate should be kept in a sealed bag to minimize exposure to damp air. Use up the reagents as soon as possible after the kit is unpacked.

 

ASSAY PROCEDURE

 

1.        Allow all reagents to reach room temperature before use.

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 two positive and two negative controls.  Dispense one drop (50 ul) of Positive Control as well as Negative Control in duplicate into respective wells. Set one black well as background control, and 50ul of serum or plasma samples into respective wells.

4.        Add one drop (50 ul) of Enzyme Conjugate to each well. Mix it gently by swirling the microtiter plate on flat bench for 1 min. Do not add Enzyme Conjugate to the blank well.

5.        Place the microtiter plate into a humidified box and incubate at 37°C for 30 min.

6.        Wash each well 5 times by filling each well with diluted wash buffer, then invert the plate vigorously to get all water out and block the rim of each well on absorbent paper for a few seconds.

7.        Add one drop (50 ul) of Substrate Solution A to each well, then add one drop (50 ul) of Substrate Solution B to each well. Mix gently and incubate at 37°C for 15 min.

 

 

 

 

 

EIA Reader at 450 nm (using the OD value of the blank well to correct all the OD reading from all wells or at 450 nm/630 nm with dual filter plate reader.

 

Cut-off Calculations:

 

Take average OD values of Negative control and times 2.1:

 

                2.1×NC x = Cut-off.

 

If the OD value of the negative control is less than 0.07, it should be reported as 0.07. If it is more than 0.07, it should be reported as the actual OD value measured.

 

 

 

Positive OD reading:  ≥Cut-off value

 

Negative OD reading: < Cut-off value