HBsAg ELISA Kit

HBsAg ELISA is used for the qualitative determination of Hepatitis B surface antigen (HBsAg) in human serum or plasma. This test is indicated for the screening of blood and blood products to be used for transfusion and an aid for the diagnosis of existing or previous hepatitis B infection.Hepatitis B is an infectious illness caused by hepatitis B virus (HBV) which infects the liver of hominoidea, including humans, and causes an inflammationcalled hepatitis. 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.
about 7.18% carry hepatitis B Virus surface antigen. about 93 million people are chronic carriers of HBV among which about 30 million are suffering Hepatitis B around the country. 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 e antigen(HBeAg)/HBeAb. As it is difficult to detect the core antigen in the serum, the other five are been done to diagnosing HBV. The surface antigen contains the determinant “a”, common to all known viral subtypes and immunologically distinguished in two distinct subgroups (ay and ad). HBV has 10 major serotypes and four HBsAg subtypes have been recognized (adw, ady, ayw, and ayr). HBsAg can be detected 2 to 4 weeks before the ALT levels become abnormal and 3 to 5 weeks before symptoms develop. The serological detection of HBsAg is a powerful method for the diagnosis and prevention of HBV infection and ELISA has become an extensively used analytical system for screening of blood donors and clinical diagnosis of HBV in infected individuals The HBsAg EIA is a solid-phase simultaneous sandwich immunoassay, which employs monoclonal antibodies and polyclonal antibodies specific for HBsAg. Microtiter well is coated with monoclonal antibodies specific for HBsAg. A serum specimen is added to the antibody coated Microtiter wells together with enzyme conjugated polyclonal antibodies. HBsAg, if present, will form an antibody-HBsAg-antibody-enzyme complex. The plate is then washed to remove unbound material. Finally, a solution of substrate is added to the wells and incubated. A blue color will develop in proportion to the amount of HBsAg present in the specimen. The enzyme-substrate reaction can be stopped and the result is visualized by naked eye or read by EIA plate reader for absorbance at the wavelength of 450 nm(450 nm/630 nm) and is proportional to the amount of antibodies present in the specimen.. REAGENTS
Item
DescriptionCoating PlateNegative ControlPositive ControlEnzyme ConjugateWash Buffer Concentrate (20x)Substrate Solution ASubstrate Solution BStop SolutionPlastic BagSeal Paper 11
Manual
It is strongly advised to analyze each specimen and controls in duplicate. All the reagents should equilibrate to room temperature before use.Dispense one drop (100μl) of Positive Control as well as Negative Control in duplicate into respective wells. Set one blank well as background control, and 100ul of serum or plasma samples into respective test wells.Place the microtiter plates into a humidified box, and incubate at 37°C for 60 minutes. Add one drop (50μl) of Enzyme Conjugate to each well. Mix it gently by swirling the microtiter plate on flat bench for 1 minute. Do not add Enzyme Conjugate to the blank well.Place the microtiter plates into a humidified box, and incubate at 37°C for 30 minutes. Wash each well 5 times by filling each well with diluted wash buffer, then inverting the plate vigorously to get all water out and blocking the rim of wells on absorbent paper for a few seconds.Add one drop (50μl) of Substrate Solution A (HRP-substrate) to each well, then add one drop (50μl) of Substrate Solution B (TMB) to each well. Mix gently and incubate at 37°C for 30minutes.