The sample is placed in contact with an anti-HBs monoclonal
antibody immobilized onto a solid support. If it contains the
HBsAg antigen, it will form a complex with the antibodies and
will bind to the support. The unbound fraction is eliminated
by washing, after which another anti-HBs monoclonal antibody conjugated to peroxidase is added. If a reaction was
produced during the first stage of the process, the conjugate
will bind. After a new wash the enzymatic substrate is added.
If HBsAg is present in the sample, a light blue color is developed. The reaction is stopped by adding sulfuric acid, which
makes the light blue color change to yellow.
Coated microtitration plate: microtitration plates with removable strips with wells containing immobilized anti-HBs
Concentrated Conjugate: anti-HBs monoclonal antibody
conjugated to peroxidase.
Conjugate Diluent: Tris buffer containing additives and
Substrate A: 60 mmol/l hydrogen peroxide in 50 mmol/l
Substrate B: 0.01 mol/l tetramethylbenzidine (TMB) in 0.1 N
Stopper: 2 N sulfuric acid.
Concentrated Wash Buffer: 1.4 mol/l sodium chloride in 100 mmol/l phosphate buffer and 0.1 g/l non-ionic surfactant.
Positive Control: dilution of inactivated serum reactive to
Negative Control: dilution of non-reactive inactivated serum.