HBeAb Antibody Rapid Elisa Test 3ml Standard Diluent Qualitative Enzyme Immunoassay

HBeAb Antibody ELISA Test detect Anti-HBe Antibody,96wells/kit, Elisa Sandwich method, for quantitative measurement

Product Name:

HBeAb Antibody ELISA Test Kit

Anti-HBe antibody ELISA Test Kit

Intended Use:

Anti-HBeAg antibody (HBeAb) EIA is a qualitative enzyme immunoassay for the detection of antibody to e antigen of hepatitis B virus (HBe) in human serum or plasma.

Summary:

The presence of antibody against hepatitis B viral e antigen is used as an indicator for (1) early HBs antigenemia before the peak of viral replication and (2) early convalescence when HBeAg has declined below detectable levels. It is also useful to confirm a seroconversion. The seroconversion from HBeAg positivity to anti-HBe positivity indicates a reduced level of infectious virus because virus replication has decreased.

Anti-HBe antibody test is a competitive enzyme immunoassay in which anti-HBe antibodies from specimens compete with a constant amount of Horseradish Peroxidase (HRP) conjugated anti-HBe antibody for a limited number of HBeAg in the neutralizing reagent added to the well. The microtiter well is coated with polyclonal antibodies against HBeAg as catcher for HBeAg. A serum specimen is added to the microtiter wells together with HRP conjugated anti-HBe antibody as well as HBeAg in Neutralizing Reagent. After incubation, anti-HBe antibodies in specimen, if present, compete with constant amount of HRP-conjugated anti-HBe for limited amount of HBeAg added into the wells. The unbound enzyme conjugates will be washed away and the chromogen substrate solution containing hydrogen peroxide is added to the wells for color development. Thus, the amount of HRP-conjugated anti-HBe bound to the well is inversely proportional to the concentration of anti-HBe antibody in the specimen. The absorbance of controls and specimens is determined using EIA reader with wavelength set at 450 nm.

Regents Provided

1.  Microtitre Coated Plate (96 wells) – 1 no

2.  Human Biotin Conjugate, 1ml – 1 vial

3.  Standard, 3200U/L, 0.5ml – 1 vial

4.  Streptavidin HRP Conjugate, - 6 ml

5.  Wash Buffer (30X) – 20ml

6.  Standard Diluent – 3ml

7.  Substrate A – 6ml

8.  Substrate B – 6ml

9.  Stop Solution – 6ml

10.  Instruction Manual

TEST PRINCIPLE

This assay is based on sandwich enzyme-linked immunosorbent assay (ELISA).Samples containing antibodies against hepatitis B virus e reacts with pre-coated antigen hepatitis B virus e in the well. After incubation, biotin labeled antigen (hepatitis B virus e) is added into the wells. Streptavidin-HRP is added to form an immune complex. After incubation, washing is done to remove the unbound complex. TMB substrate is added to the wells. The amount of hydrolyzed substrate is read on a microplate reader and it is directly proportional to the concentration of antibodies against hepatitis B virus E.

TEST PROCEDURE

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

2.  Dispense one drop (50 ul) of specimen, Positive Control as well as Negative Control into respective wells.

3.  Add one drop (50 ul) of Enzyme Conjugate to each well, followed by one drop (50 ul) of Neutralizing Regent. Mix them gently by swirling the microtiter plate on flat bench for 2 min.

4.  Place the microtiter plate into a humidified box and incubate at 37o C for 60 min.

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

6.  Add 1 drop (50 ul) of Substrate Solution A to each well, then add 1 drop (50 ul) of Substrate Solution B to each well. Mix gently and incubate at room temperature for 15 min.

7.  Visual inspection of the color reaction in each well or add one drop (50 ul) of Stop Solution to each well to stop the color reaction. Blank EIA reader with a blank control well and then read O.D. values of all samples at 450 nm.

Performance Characteristics:

Please note that this validation is performed in our laboratory and will not necessarily be duplicated in your laboratory. This data has been generated to enable the user to get a preview of the assay and the characteristics of the kit and is generic in nature. We recommend that the user performs at the minimum; the spike and recovery assay and the dilutional linearity assay to assure quality results. For a more comprehensive validation, the user may run the protocols as suggested by us herein below to develop the parameters for quality control to be used with the kit.

Sensitivity: Limit Of Detection: It is defined as the lowest detectable concentration corresponding to a signal of Mean of ‘0’ standard plus 2* SD. 10 replicates of ‘0’ standards were evaluated and the LOD was found to be 10 U/L.

Precision:

Intra-assay Precision: 3 samples with low, middle and high level Human HBeAb were tested 20 times on one plate, respectively.

Inter-assay Precision: 3 samples with low, middle and high level Human HBeAb were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/mean x 100 Intra-Assay: CV<10% Inter-Assay: CV<12%

Ver1.0 5