HBSAg Elisa test:

Assay procedure:

1.    Bring all the reagents to room temperature
2.   Arrange required no. of wells and proceed
3.   100µl of negative control11 (R3) in well1A1.B1.C1 &D1
4.   100 µl of HBSAg positive control (R4) in well E1
5.   100 µl of test sample to other remaining wells
6.   50 µl of working conjugate to each well (Working conjugate = Mix conjugate-R7+ conjugate diulent-R6) Mix well, seal with adhesive film and incubate at 37° C for 1.30 hours
7.   Wash 5 times with working wash Solution (Concentrate - R2 100 ml + Dist. Water 1000 ml)
100 µl fresh working development solution
(Chromogen-R9 1ml+ Substrate buffer R810 ml) Incubate for 30 minutes at dark place. 100 ul stopping solution (ready to use)
 Read at 450 & 620 after 4 minutes Calculation

Calculation

Cut- off value = Mean absorbance O.D of Negative control + 0.050
Calculation of ratio sample = O.D. sample / Cut-off value

Sample ratio less than 1 are considered negative, sample ratio equal or
greater than 1 is considered as positive
Assay validation

 Negative control         :         < 0.080

 Positive control  :         > 1.000

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