For the Visual Determination of Hepatitis B Surface Antigen (Hbsag) Concentration in Human
HBsAg , Page 1
MICROWELL ELISA
HBsAg
Catalog No. 1701
(96 tests)
INTENDED USE
For the Visual determination of Hepatitis B Surface Antigen (HBsAg) concentration in human serum.
INTRODUCTION
Infections by Hepatitis B Virus (HBV) occur, and present serious public health problems in all parts of the world. Because HBV infections are widely spread, early detection of the infection is essential. A variety of serological markers appear following infection with HBV, and one of first of these markers is Hepatitis B Surface Antigen (HBsAg). This antigen appears before biochemical evidence of liver disease or jaundice, persists through the acute disease phase, and declines during convalescence.
PRINCIPLE OF THE TEST
The HBsAg Visual Test Kit is based on a solid phase enzyme-linked immunosorbent assay. The assay system utilizes one anti-HBsAg antibody for solid phase (microtiter wells) immobilization and another Goat anti-HBsAg antibody in the antibody-enzyme (horseradish peroxidase) conjugate solution. If HBsAg is present in the specimen, it will combine with the antibody on the well and the enzyme conjugate resulting in the HBsAg molecules being sandwiched between the solid phase and enzyme-linked antibodies. After a 45 minutes incubation at 37C, the wells are washed with water to remove unbound labeled antibodies. A solution of TMB is added and incubated for 30 minutes, resulting in the development of a blue color. The color development is stopped with the addition of 2N HCl. The color is changed to yellow and measured spectrophotometrically at 450 nm. The concentration of HBsAg is directly proportional to the color intensity of the test sample.
Upon completion of the test, color development suggests the presence of HBsAg; the absence of color suggests the absence of HBsAg.
MATERIALS AND EQUIPMENT REQUIRED
Materials provided with the test kits:
Antibody coated microtiter wells, 96 wells per plate.
Enzyme Conjugate Reagent, 6 ml.
Positive Control, 1 ml / vial , (Red Color,Ready for use.)
Negative Control, 1 ml / vial , (Yellow Color,Ready for use.)
- Wash Buffer (20X), 30 ml .
Color Reagent A, 6 ml.
Color Reagent B, 6 ml.
- Stop Solution, 6 ml
Materials required but not provided:
Distilled water.
SPECIMEN COLLECTION
1.Blood should be drawn using standard venipuncture techniques and the serum should be separated from the red blood cells as soon as practical. Avoid grossly hemolytic, lipidic or turbid samples.
2.Plasma samples collected in tubes containing EDTA, heparin, or oxalate may interfere with test procedures and should be avoided.
3.Specimens should be capped and may be stored for up to 48 hour at 2-8C prior to assaying. Specimens held for a longer time can be frozen at -20C for mix prior to testing.
STORAGE
Unopened test kits should be stored at 2-8C upon receipt and the microtiter plate should be kept in a sealed bag with desiccants to minimize exposure to damp air.
REAGENT PREPARATION
1.All reagents should be brought to room temperature (18-25C ) before use.
2.Gently swirl each bottle liquid reagent. Do not shake or agitate reagent bottle vigorously.
3. Dilute 0.5 volume of Wash Buffer (20x) with 9.5 voumes of distilled water. For example, Dilute 15 ml of Wash Buffer (20x) into distilled water to prepare 300 ml of wash buffer(1x). Mix well before use.
ASSAY PROCEDURE
1.Secure the desired number of antibody-coated wells needed for the samples and controls to be tested.
2.Using a dispensing pasture pipet, add 50 ul of Negative control, 50 ul of Positive Control and 50 ul of each patient sample to the wells.
3.Add 50 l (one drop) of the Enzyme Conjugate Reagent to each well. Shake the microtiter plate gently and incubate at 37C for 45 minutes.
4.Remove the incubation mixture by flicking plate content into sink. Rinse and flick the microtiter plate four times with wash buffer (1x) and one time distilled water. Strike the microtiter plate sharply onto absorbent paper or paper towels to remove all residual water droplets.
5.Add 50 l (one drop) of Color Reagent A to each well, followed by 50 l (one drop) of Color Reagent B. It is important that Color Reagent A is added to each well before Color Reagent B.
6.Mix gently and incubate at 37C for 30 minutes.
7.Stop the reaction by adding 50 l (one drop) of Stop Solution. Read the results at 450nm.
8.Compare the color of the patient sample wells to the color of the Negative and Positive Control wells.
Important note:
The wash procedure is critical. Insufficient washing will result in improper color development.
INTERPRETATION OF RESULTS
Cut-Off Value
The cut-off value equals to two times of the OD reading of the negative control. For exsample, if the negative control OD reading is 0.070, the cut-off value will be 0.070 x 2 = 0.140
Positive
The positive well should develop a distinct blue color, and the OD reading should be larger than the cut-off value.
Negative
Samples producing no blue color should be considered negative, and the OD reading should be less than the cut-off value.
RELATED READING MATERIALS
- Zuckerman, AJ. In immunoassays for the 80’s. A. Voller, A. Bartlett, and D. Bidwell (eds.) University Park Press, Baltimore, 1981: 361-373.
- Wolters G. Kuijpers, LPC and Schuurs AHWM Hepatitis Scientific Memoranda, 1975: 908-911
Revised 5/99
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