1. HbsAg SCREENING
HbsAg ELISA test for the detection of hepatitis B surface
Antigen (HbsAg) in human serum or plasma
1. The provided micro plate has 12 columns (strips) and 8 rows (A-H).
2. Make a summary sheet of the micro plate.
3. A1 is for Blank, B1, C1, D1 are for Negative Controls (100ul each), E1, F1 are for
Positive Controls (100ul each).
4. G1 onwards are test samples.
5. Seal plate with the adhesive tape (provided with kit).
6. Incubate at 37 C for 60 minutes.
7. Wash plate with Washing Solution 5 times. (to be diluted 1/10 in distilled water i.e. 100ml
Washing Solution in 1000 ml Distilled Water)
8. Concentrate Conjugate to be diluted 1/51 with Conjugate Diluent i.e. 20ul concentrate
conjugate in 1ml Conjugate Diluent conjugate. (For 1 strip ___ 1ml diluent conjugate,
similarly in 6ml diluent conjugate + 120ul concentrate conjugate).
9. Put 100ul of above in each well except the Blank well and seal.
10. Incubate at 37 C for 30 minutes.
11. Wash plate 5 times with Washing Solution.
12. Make Substrate reagent (1ml/strip, add 20ul/ml Conc. Substrate with diluting solution).
13. Put 100ul of above in each well including Blank well.
14. Incubate at 22 C for 30 minutes (in dark at the bench).
15. Color develops. Add stopping solution (N/10 H2SO4).
16. Take readings as Negative or Positive.
Blood Transfusion Services
Pakistan Institute of Medical Sciences
051-9261272, 051-9261079 Ext; 2542
1

2. HCV ANTIBODIES TEST
Third generation ELISA test for the detection of antibodies
to HCV in human serum or plasma.
1. The provided micro plate has 12 column (Strips) and 8 rows (A-H).
2. Make a summary sheet of the micro plate.
3. A1 is for blank, B1, C1, D1, are for NEGATIVE CONTROLS (200ul each), E1, F1 are for
POSITIVE CONTROLS (200ul each).
4. G1 onwards are Test samples.
5. Put 200ul of SAMPLE DILUENT in each samples well (and not in the control samples).
6. Add 10ul of donor Test samples in respective wells.
7. Seal the plate with the provided adhesive tape and incubate the plate at 37 C for 60
minutes in Incubator Shaker.
8. Wash plate 5 times with WASHING SOLUTION (100ml washing solution diluted with
1000ml of distilled water).
9. Make Conjugate Solution as Table 1.
10. Add 100ul of above diluted Conjugate in each well accept Blank well.
11. Seal the plate with adhesive tape and incubate the plate at 37 C for 30 minutes in the
Incubator Shaker.
12. Wash plate 5 times with WASHING SOLUTION.
13. Make SUBSTRATE SOLUTION also called TMB or Chromogen as in Table 2
14. Add 100ul of above TMB Solution in each well.
15. Incubate the plate at room temperature (22 C) for 30 minutes in dark.
16. Add 100ul Stop Solution (N/10 H2SO4) in each well.
17. Take reading as Negative or Positive.
Blood Transfusion Services
Pakistan Institute of Medical Sciences
051-9261272, 051-9261079 Ext; 2542
2