RUO HBcAb Elisa kit

The purpose of the RUO HBcAb ELISA Test is intended for the qualitative detection of antibodies to hepatitis B virus Core antigen in human serum/plasma.

As part of the Hepadnaviridae family, HBV is an enveloped, double-stranded DNA virus that is a primary cause of hepatitis transmission through blood. The effects of HBV infection range anywhere from mild to severe hepatitis, which includes chronic liver problems, such as carcinoma and cirrhosis. In order to classify hepatitis B infection, the serological markers need to be identified during the three phases of the infection - incubation, acute, and convalescent. The main component of the virus is Hepatitis B core antigen (HBcAg). This core antigen is comprised of a single polypeptide of approximately 17kD that is discharged upon disaggregation of the core particles. At least one immunological determinant is present in the antigen. Shortly after the onset of HBsAg, antibodies to HBcAg (anti-HBc total antibody and IgM) appear and never go away. In isolated cases, a Hepatitis B infection can be contracted without immunologically detectable anti-HBc. This is found usually in immunosuppressed patients. Screening for anti-HBc yields data on the prevalence of hepatitis B in various populations. This is because anti-HBc is a marker of acute, chronic, or resolved HBV infection. The identification of anti-HBc is vital when being diagnosed in a clinical setting. Together with other hepatitis B tests, the anti-HBc marker allows correct diagnosis and proper monitoring of progress of the virus. Anti-HBc may possibly be the only indicator of a hepatitis B infection (including other tests of HBsAg-negative patients).

MATERIALS AND COMPONENTS

MICROPLATE: Blank microwell strips fixed on white strip holder. The plate is sealed in aluminum pouch with desiccant. Each well contains purified HBcAg. The microwell strips can be broken to be used separately. Place unused wells or strips in the provided plastic sealable storage bag together with the desiccant and return to 2-8°C. Once open, stable for one month at 2-8°C.

NEGATIVE CONTROL: (1x1.0ml per vial) preserv.0.1% ProClinTM 300 Yellowish liquid filled in a vial with green screw cap.Protein-stabilized buffer tested non reactive for HBcAb. Ready to use as supplied. Once open, stable for one month at 2-8°C.

POSITIVE CONTROL: (1x1.0ml per vial) preserv.0.1% ProClinTM 300 Red-colored liquid filled in a vial with red screw cap. HBcAb diluted in protein-stabilized buffer. Ready to use as supplied. Once open, stable for one month at 2-8°C.

CONJUGATE: (1x6ml per vial) preserv.0.1% ProClinTM 300 Red-colored liquid in a white vial with red screw cap. Horseradish peroxidase-conjugated monoclonal antibodies to HBcAg. Ready to use as supplied. Once open, stable for one month at 2-8°C.

WASH BUFFER: (1x20ml per bottle) DILUTE BEFORE USE! detergent Tween-20 Colorless liquid filled in a white bottle with white screw cap.PH 7.4, 20 × PBS The concentrate must be diluted 1 to 19 with distilled/deionized water before use. Once diluted, stable for one week at room temperature, or for two weeks when stored at 2-8°C.

SUBSTRATE A: (1x6ml per vial) Colorless liquid filled in a white vial with green screw cap. Urea peroxide solution. Ready to use as supplied. Once open, stable for one month at 2-8°C.

SUBSTRATE B: (1x6ml per vial) Colorless liquid filled in a black vial with black screw cap.TMB (Tetramethyl benzidine) solution. Ready to use as supplied. Once open, stable for one month at 2-8°C.

STOP SOLUTION: (1x6ml per vial) Colorless liquid in a white vial with white screw cap.Diluted sulfuric acid solution (0.5M H₂SO₄).Ready to use as supplied. Once open, stable for one month at 2-8°C.

PLASTIC SEALABLE BAG: For enclosing the strips not in use 1unit

PACKAGE INSERT 1copy

CARDBOARD PLATE COVER 2sheets

To cover the plates during incubation and prevent evaporation or contamination of the wells.

PROCEDURE

Reagents preparation: Allow the reagents to reach room temperature (18-30°C). Dilute the Wash buffer (20X) as indicated in the instructions for washing. Use distilled or deionized water and only clean vessels to dilute the buffer. All other reagents are READY TO USE AS SUPPLIED.

1. Preparation: Format the microplate’s wells for control and patient specimen to be assayed. Replace any unused microwell strips back into the aluminum bag seal and store at 2-8°C.
2. Adding Sample: Add 50μl of the control or the samples into the assigned well.
3. Adding Conjugate: Add 50μl of CONJUGATE into each well except the Blank.
4. Incubating: Cover the plate with the plate cover and incubate for 30 minutes at 37°C.
5. Washing: At the end of the incubation, remove and discard the plate cover. Wash each well 5 times with diluted Wash Buffer. Each time allow the microwells to soak for 30-60 seconds. After the final washing cycle, turn down the plate onto blotting paper or clean towel and tap it to remove any remainders.
6. Adding Substrate: Add 50μl of Substrate Solution A and 50μl of Substrate Solution B into each well. Incubate for 10 minutes at 37°C avoiding light.
7. Adding Stop Solution: Using a multichannel pipette or manually, add 50μl of Stop Solution into each well and mix gently.
8. Measuring the Absorbance: Calibrate the plate reader with the Blank well and read the absorbance at 450nm. If a dual filter instrument is used, set the reference wavelength at 630nm. Calculate the Cut-off value and evaluate the results. (Note: read the absorbance within 10 minutes after stopping the reaction).