Hepatitis B Surface Antigen Elisa Test Kit , Elisa Diagnostic Kits In Human Serum

HBsAb ELISA test detect Hepatitis B surface antigen,96wells/kit, Elisa Sandwich method, for quantitative measurement
 
 
 
Intended Use:
 
 
The HBsAb ELISA Test is a solid phase enzyme linked immunoabsorbent assay for the qualitative detection of antibodies (IgG, IgM, IgA) against hepatitis B virus surface antibody in human serum or plasma. It is intended for professional use only as an aid in the diagnosis of infection with HBV. Any reactive specimen with the HBsAb ELISA Test must be confirmed with alternative testing method(s) and clinical findings.
 
Summary:
 
Hepatitis B virus is a spherical enveloped, partially double-stranded DNA virus of the Hepadnaviridae family.1 The Hepatitis B infection of the liver is transmitted through sexual contact, blood borne exposure, transmission from mother to child during delivery, sharing of objects that pierce the skin, child-to-child and household contact.2,3,4,5 HBV infection has been linked to a variety of mild to chronic liver diseases, including cirrhosis, and hepatocellular carcinoma.4 In some cases, the virus may persist for a lifetime. Annually, 1 million people die from chronic active hepatitis, cirrhosis or primary liver cancer. Hepatitis B affects millions of people worldwide and is considered a global public health problem.4,5 HBsAg is one of the earliest markers that appear in the blood following infection with the Hepatitis B virus. The body’s immune response to infection includes the development of specific antibodies to HBsAg. These antibodies appear a few weeks after HBsAg is cleared from the blood and the virus can no longer be passed on to others. The appearance of HBsAb is associated with recovery and is regarded as the marker for immunity. These antibodies also appear as a result of successful immunization.3,5 Therefore, the detection and monitoring of antibodies to HBsAg has become an important tool in the screening and monitoring of infected individuals as well as an indicator of those successfully vaccinated against HBV. The HBsAb EIA Test Kit is an immunoassay for the qualitative detection of the presence of Hepatitis B Surface Antibody (HBsAb) including IgG, IgM and IgA antibodies in serum or plasma specimen.
 
Regents Provided
 
1. Microtitre Coated Plate (96 wells) – 1 no
2. Human Biotin Conjugate, 1ml – 1 vial
3. Standard, 280IU/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
 
The HBsAb EIA Test Kit is a solid phase qualitative enzyme immunoassay based on the sandwich principle for the detection of HBsAb including IgG, IgM and IgA antibodies in human serum or plasma. The microwell plate is coated with recombinant HBsAg. During testing, the specimens are added to the antigen coated microwell plate and then incubated. If the specimen contains HBsAb, it will bind to the antigen coated on the microwell plate and simultaneously bind to the conjugate to form immobilized antigen-HBsAb-conjugate complexes. If the specimen does not contain HBsAb, the complexes will not be formed. After initial incubation, the microwell plate is washed to remove unbound materials. Substrate A and substrate B are added and then incubated to produce a blue color, indicating the amount of HBsAb present in the specimen. Sulfuric acid solution is added to the microwell plate to stop the reaction which produces a color change from blue to yellow. The color intensity, which corresponds to the amount of HBsAb present in the specimen, is measured with a microplate reader at 450/630-700 nm or 450 nm
 
TEST PROCEDURE

1. Bring all reagents to room temperature prior to use. It is strongly recommended that all Standards and Samples should be run in duplicates or triplicates. A standard curve is required for each assay.
2. Standards Dilution: Prepare the standards as per the table given below using the provided standard concentration and standard diluent.

1. Remove the number of strips required for the assay.
2. Pipette out 50µl of Standards and Samples into the respective wells as mentioned in the work list. Note do not add the sample to the blank well.
3. Pipette out 50µl of Biotin Conjugate into each sample well. Do not pipette into the blank and standards wells.
4. Pipette out 50µl of Streptavidin-HRP Conjugate into each sample and standards well. Do not pipette into the blank well.
5. Cover the plate and incubate for 1 hour at room temperature (37°C).
6. Aspirate and wash plate 4 times with 1X Wash Buffer and blot residual buffer by firmly tapping plate upside down on absorbent paper. Wipe off any liquid from the bottom outside of the microtiter wells as any residue can interfere in the reading step. All the washes should be performed similarly.
7. Add 100µl of TMB Substrate in each well.
8. Incubate the plate at Room Temperature for 10 minutes in dark. DO NOT SHAKE or else it may result in higher backgrounds and worse precision. Positive wells should turn bluish in color.
9. Pipette out 50µl of Stop Solution. Wells should turn from blue to yellow in color.
10. Read the absorbance at 450 nm blanking on the zero standard.

 
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:
 

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 0.856 IU/L.
 

Precision:
 

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

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