a) The different key component effects for ELISA detection
- Antibody: The antibody is the major factor determining the sensitivity and specificity of an assay. Briefly, it is the three dimensional configuration of the antigen-binding site found in the F(ab) portion of the antibody that controls the strength and specificity of the interaction with antigen. The stronger the interaction, the lower the concentration of antigen that can be detected (other factors being equal). A competing factor is the specificity of binding or the cross-reactivity of the antibody to serum proteins other than the target antigen. Depending on whether the antibodies being used are polyclonal or monoclonal, cross-reactivity will be caused by different forces. In either case driving the assay to the limit of sensitivity may result in cross-reactivity, and one is faced with the conflicting needs of sensitivity versus specificity.
b) Different standards (from different expression systems)
c) Different pairs in the kit (the antibody is Mab or Pab).
d) affinity and specificity of the antibody
e) Blocking buffer
f) chromogenic agent
g) rabbit monoclonal antibody VS other monoclonal antibody