A competitive immunoassay technique is used. This involves the reaction of anti-HBc in the sample with hepatitis B core antigen (HBcAg) coated wells. Unbound material is removed by washing. Horseradish peroxidase (HRP)-labeled antibody conjugate (mouse monoclonal anti-HBc) is then allowed to react with the remaining exposed HBcAg on the well surface.
Unbound conjugate is removed by washing. The bound HRP conjugate is measured by a luminescent reaction. A reagent containing luminogenic substrates (a luminol derivative and a peracid salt) and an electron transfer agent, is added to the wells. The HRP in the bound conjugate catalyzes the oxidation of the luminol derivative, producing light. The electron transfer agent (a substituted acetanilide) increases the level of light produced and prolongs its emission. The light signals are read by the system. The amount of HRP conjugate bound is inversely proportional to the concentration of anti‑HBc present.
Reaction scheme and results screenshot has been attached.
How is cut-off determined and 1.2 and
You're right -- the "competition" is happening at separate steps and is irreversible. This is not how a chemist would normally use the word "competition", but it's established jargon for immuoassays that work this way.
First, the sample Anti-HBc blocks all the binding sites it can, depending on how much of it there is. Then you wash. The Anti-HBc stays stuck no matter how hard you wash (irreversible). Then you detect how many sites remained open with your labeled anti-HBc.
The cutoff numbers are simply empirical, based on the expected background and sample staining intensity of your particular kit.
Why not run a simple competitive immunoassay? First titrate the binding of HRP-anti-HBc to the antigen coated wells. Establish the concentration giving you around 50% signal. Next , run the assay with co-incubation of the test sample with the HRP-anti-HBc at 50% signal and work out the % inhibition. Relate that to concentration either by running standards or sample with known concentrations of anti-HBc. Otherwise, establish your non-specific signal by using antibody free samples and then workout the cut off based on your signal/noise variability. For more sensitivity. instead of co-incubation do step incubation. first with your sample, wash then with the HRP-anti-HBc
The assay principle is that tested antigen and enzyme-labeled antigen competitively bind to the immobile antibody. The higher concentration of antigen in the tested sample, the less enzyme-labeled antigen binds to the immobile antibody, so the lower intensity of color develops after the addition of the substrate.
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