STANDARD OPERATING PROCEDURE: HBsAg TESTING BY ELISA

                                        BLOOD CENTRE NAME

SOP Number	Effective date	Pages	Author	Authorised by
Version	Review Period	No. of copies	Reviewed by	Date

LOCATION    TTI Testing Laboratory	SUBJECT   HBsAg Testing
FUNCTION           -Samples tested for Hepatitis B -Surface Antigen by ELISA method	DISTRIBUTION -Supervisor in charge of TTI Testing Laboratory – Master File

1. SCOPE & APPLICATION

HBsAg is a mandatory test for blood unit screening before it is transfused. This is carried out on all donor units’ samples.

2. RESPONSIBILITY

It is the responsibility of technician from TTI Testing lab to carry out the test and report as required.

• REFERENCE

1. Kit Package inserts.

1. Technical Manual of American Association of Blood Banks 20^th edition 2020

• MATERIALS REQUIRED

• Elisa Reader
• Elisa Washer
•  Microshaker
•  Incubator

•  Micropipettes and disposable tips
•  Timer

•  Disposable gloves

•  Disposal container with Na Hypochlorite
•  Absorbant tissue

•  Distilled water

•  1 mol / litre Sulphuric acid
•  Kit

5. PROCEDURE

5.1       Principle:

In the monoclonal EIA procedures microplate wells are coated with monoclonal antibody to Hepatitis B Surface Antigen (Anti HBs) are incubated with serum or plasma and Anti-HBs peroxidase (Horse radish) conjugate in one step assay. During the incubation period HBsAg if present is bound to the conjugate (Anti-HBs-HRPO). Unbound material is aspirated and washed away. On the addition of substrate colour develops in proportion to the amount of HBsAg which is bound. The enzyme reaction is stopped by the addition of stopping solution.

5.2       Method:

1. Carry out the test as per manufacturer’s instructions given in the package insert.
   1. Remove reagents from the fridge 30 minutes prior to testing. Mix the reagents gently by inverting the vials without foaming.
   1. Bring reagents and samples to room temperature before testing.

• Arrange all donor unit test tube samples, apheresis samples, serially in ascending order in a test tube rack. Add required number of internal kit controls and external lab controls.

• Discard all disposable tips into hypochlorite solution.

• Place the tray in front of the test tube rack.

• Dispense test sample 100 l volume using an auto pipette and fresh disposable tip for every sample.
  • Add kit internal positive control.

• Add kit internal negative control.

• Add Lab. External negative control.

• Add Lab. External strong positive control.

• Add Lab. External weak positive control.

• Agitate using microshaker speed 900 rpm for 15 seconds

• Incubate at 37⁰C for 90 minutes.

• Wash and soak each well four times with PO4 buffer. Check buffer before use. If salt crystals have formed, resolubilise by warming at 37⁰C until crystals dissolve.

• Pipette 100 l substrate in each well. Do not mix or agitate discard any unused substrate.
  • Incubate at 5-300 for 30 minutes.

• Stop reaction by adding 100 l 1 mol/L Sulphuric Acid to each well.

• Ensure thorough mixing by tapping side of plate

• Read the plates within 15 minutes

• Blank the reader or air and read the absorbance in each well at 450 +5nm.

5.3       Validation:

If the run fails to meet criteria as per package insert consider the test is invalid and repeat the whole test again. Examine absorbance values of the controls before the sample results can be interpreted.

Check the validity of the blank (if used) Negative and positive control absorbance value as per package insert of the kit.

Cut off O.D. is automatically calculated.

5.4       Interpretation:

Check the printout carefully for absorbance value:

• The samples below the cut off are considered non-reactive.

• Equal to cut off are considered initial reactive

• Above cut off are considered initial reactive

• Those with asterik are in grey zone. Repeat all samples showing grey zone result.

6. DOCUMENTATION

6.1

Paste the printout in the HBsAg Register and also record the following details:

• The date on which the test is run.

• The name of the kit used.

• Lot No. and expiry date of the kit.

• Initials of the technologist who performed the test.

• Initials of the Supervisor who verifies the result.

• Reactive units are marked in red.

6.2

Transfer the results to donor grouping register.

7. END OF DOCUMENT