HBsAg ELISA (Blood Banking)

HBsAg Microwell ELISA is an in vitro enzyme immunoassay for the qualitative detection of Hepatitis B surface antigen (HBsAg) in human serum or plasma. It is based on a direct, non-competitive, solid-phase enzyme immunoassay principle using horseradish peroxidase (HRP) as the marker enzyme. After incubation and wash steps, colour develops using TMB substrate, the reaction is stopped, and absorbance is read at 450 nm (reference 630 nm) within 15 minutes of adding stop solution (as per IFU).

For general background on hepatitis B and HBsAg testing, see:

WHO: Hepatitis B fact sheet  

CDC: Clinical testing and diagnosis for hepatitis B  

• Pack size: 96 tests
• Storage (kit): 2°C to 8°C
  
• Expiry : 18 months

Key features

• Qualitative detection of HBsAg in human serum or plasma
• Direct, non-competitive, solid-phase microwell ELISA principle
• Read at 450 nm with 630 nm reference wavelength
• Includes positive and negative controls
• Analytical sensitivity 0.4 PEIU/ml, Diagnostic sensitivity 100%, Diagnostic specificity 100%

Intended use

HBsAg Microwell ELISA is an in vitro enzyme immunoassay for the qualitative detection of Hepatitis B surface antigen in human serum or plasma.

Principle of the test

HBsAg Microwell ELISA is a direct, non-competitive, solid-phase enzyme immunoassay with HRP as the marker enzyme. In brief:

1. If present, HBsAg in the sample binds to anti-HBs antibodies coated on the microwell.
2. An HRP-conjugated anti-HBs antibody binds to the complex.
3. After washing, TMB substrate produces a colour change; the reaction is stopped and absorbance is measured.

Specimen requirements and storage

• Specimen: Human serum or plasma
• Store specimens at 2–8°C.
• If specimens are not tested within 7 days, store frozen at -15°C or colder.
• Avoid multiple freeze–thaw cycles. After thawing, ensure specimens are thoroughly mixed before testing.

(For clinical testing guidance and interpretation frameworks, refer to national/public health guidance such as the CDC’s hepatitis B testing page.) 

Cut-off value and interpretation of results

Cut-off value (COV):

1. Calculate the negative control mean (NCx)
2. COV = NCx + 0.100

Reactive: Sample absorbance ≥ COV

Non-reactive: Sample absorbance < COV

Grey zone: Absorbance within ±10% of COV

Retesting note : If samples are considered reactive, the test should be conducted two more times.

Quality control (assay validity criteria)

Results are valid if the following control criteria are met:

• Blank absorbance < 0.050
• Mean absorbance of negative controls < 0.100
• Mean absorbance of positive controls > 1.000

If results fall outside these ranges, the test should be conducted again.

Performance characteristics (as stated in the IFU)

• Analytical sensitivity: 0.4 PEIU/ml
• Diagnostic sensitivity: 100%
• Diagnostic specificity: 100%
  
  

Kit contents (96 tests)

1. HBsAg antibody coated microstrips (1 × 96 wells)
2. Sample diluent (1 × 6.0 ml)
3. Negative control (1 × 0.5 ml)
4. Positive control (1 × 0.5 ml)
5. Conjugate concentrate (101x) (1 × 0.3 ml)
6. Conjugate diluent (1 × 15 ml)
7. TMB substrate concentrate (101x) (1 × 0.3 ml)
8. Substrate buffer (1 × 15 ml)
9. Wash solution concentrate (20x) (1 × 30 ml)
10. Stop solution (1 × 6 ml)
11. Microstrip covers (02 nos.)
12. Product insert (01 no.)
13. Procedure card (01 no.)

Microplate handling note : Allow wells to reach room temperature (18–30°C), return unused wells to 2–8°C, and once opened, microwells should be used within one month.

Precautions 

• Handle specimens as potentially infectious; use appropriate PPE.
• Do not use reagents beyond the stated expiry date.
• Do not modify the test procedure or substitute reagents unless stipulated as interchangeable.
• Do not allow wells to become dry during the assay procedure.

(Refer to the IFU for the full precautions list.)

FAQs

What is the HBsAg ELISA test used for?

It is an in vitro enzyme immunoassay for the qualitative detection of Hepatitis B surface antigen (HBsAg) in human serum or plasma.

What sample type is required?

Human serum or plasma.

How are results interpreted?

A cut-off value (COV) is calculated as NCx + 0.100. Samples with absorbance ≥ COV are reactive; < COV are non-reactive; values within ±10% of COV are grey zone.

At what wavelength is the test read?

Absorbance is read at 450 nm with 630 nm as reference wavelength.

What are the performance characteristics stated in the IFU?

Analytical sensitivity is 0.4 PEIU/ml, with diagnostic sensitivity 100% and diagnostic specificity 100% (as stated in the IFU).

What is the shelf life of the kit?

Refer to the kit label for the specific lot’s manufacturing and expiry dates.

For general information about hepatitis B, see WHO’s overview.