MLS203 Hepatitis B ELISA Protocol.

HBsAg ELISA procedure:

Materials and reagents required

 2 x Microwell strips (containing 8 wells  Micropipettes and tips (100 ul)

each)
 Sample diluent (green/brown buffer)(SD)
 Negative control (NC)
 Positive controls (PC)
 Conjugate Solution (red solution) (CS)
 Working Substrate Solution (Pink solution)
(WSS)
 Stopping solution (colourless solution)
(STOP)
 Samples (S1, S2, S3, S4)
 Dry heat incubator (37 degrees)
 Plate washer and reader
 70% ethanol & Bleach to disinfect
benches
 Paper towels (for blotting)
 Timer
 Gloves

Procedure:
1. Perform equipment maintenance and calibration, where necessary, as required by
the manufacturer (this has already been done for you).
2. Bring all the reagents to room temperature before beginning the assay procedure.
3. Mix reagents by inverting a few times just before use.
4. You will be provided with 2 microwell strips containing 8 wells each
5. Use only the number of wells required for the test.
6. Identify the individual wells for each control or specimen -run in duplicates. The
layout of samples and controls is shown on attached Micro-well Plate map.
7. Add 25µl of Sample diluent to each well
8. Add 75ul of samples or controls to the well according to the map attached. Mix by
pipetting up and down gentle.
9. Cover the microwell plate with a plate sealer or use other means to minimize
evaporation. Incubate the plate for 30 ± 5 minutes at 37 ± 2°C.
10. Add 50ul of conjugate to each well
11. Shake the plate using a plate shaker for 10sec or manually agitate by gentle tapping
the sides for 10 sec.
12. Cover the microwell plate and incubate the plate for 15 ± 5 minutes at 37 ± 2°C.
13. At the end of the incubation period, carefully remove the plate cover and wash the
plate by filling the wells with 500ul wash buffer followed by aspiration of the wash
buffer. Wash 5 times. In this practical session we will use a microplate washer. After
the last wash, invert the plate and tap out any residual wash fluid into the absorbent
paper. NOTE: Grasp the plate holder firmly along the long sides before inverting to
blot (this will be demonstrated to you).
 14. Immediately after washing the plate, add 100ul of working substrate solution to
each well.
15. Cover the microwell plate and incubate the plate for 15 ± 5 minutes at 37 ± 2°C
while colour develops. A purple colour should develop in wells containing reactive
samples.
16. Add 50ul of stop solution to each well.
17. Read absorbance within 15 minutes after adding the stopping solution, using the
450 nm filter. Ensure that all strips have been pressed firmly into place before
reading. (This will be demonstrated to you).

Micro-well Plate map

1 2 3 4 5 6 7 8 9 10 11 12

A NC NC

B PC- PC-
HBsAg HBsA
g
C S1 S1

D S2 S2

E S3 S3

F S4 S4

Assay validation
- A run is valid if NC mean absorbance is less than 0.2
- A run is valid if PC mean absorbance is 0.8 above NC mean
- If any of these criteria have not been met, the assay is invalid and must be repeated
- If the run is valid calculate the cut-off value.
Cut-off value= Mean NC + 0.05
Interpretation of results:
- The presence or absence of HBsAg is determined by relating the
absorbance value of the sample to the cut-off value.
- Samples with absorbance values less than the cut-off value are
considered non-reactive (NEGATIVE) Further testing is not required.
- Samples with absorbance values greater than or equal to the
Cut-off value are considered reactive initially and should be retested in duplicate to
validate the initial test results. (For the purposes of this lab, you will not repeat the
results……report these as POSITIVE)