Hello Brandon Hanna

In a sandwich ELISA, higher signals in non-diseased control samples compared to diseased samples could be because of the hook effect in diseased samples. When there is an excessive amount of antigen in a diseased sample, the antigen saturates all available binding sites on both the capture and detection antibodies. This prevents the formation of the antibody-antigen-antibody sandwich structure, leading to a falsely low or negative signal. The non-diseased controls, with a lower or zero antigen concentration, would not experience this effect and would produce a normal, and in comparison, higher signal.

To check for the hook effect, perform serial dilutions of your diseased serum samples. If the signal of the diseased samples increases at higher dilutions, you have confirmed the hook effect. The extremely high concentration of the antigen in the diseased sample interferes with the immunoassay's ability to form stable immune complexes, leading to an artificially low result. Diluting the sample reduces the antigen concentration, allowing the antibodies to bind properly and form a stronger signal, thus confirming the hook effect.

Secondly, there may be interference from serum components. Complex samples like serum contain numerous proteins, lipids, and other substances that can interfere with the ELISA. If the non-diseased controls have a higher concentration of these interfering antibodies than the diseased samples, they will show a higher signal.

Finally, the detection antibody might have some non-specific binding affinity for other proteins in the non-diseased serum matrix. This would generate a higher background signal in the control group compared to the diseased samples. Also, there could be a possibility that the disease state might alter the target antigen's conformation, masking or modifying the epitope recognized by one or both antibodies. If the target antigen in the diseased samples is structurally different, it might not be properly "sandwiched," causing a low signal, while the normal controls with the correct antigen conformation will yield a higher signal.

You may want to follow the below steps to solve this problem.

1. First and foremost, create a dilution series of both your control and diseased serum samples. If the signal of the diseased samples increases at higher dilutions, you have confirmed the hook effect.

2. To test for interference, run a negative control using a sample matrix (e.g., buffer without serum, or serum depleted of the target antigen) to establish the true background level.

3. Optimize your blocking buffer and sample diluent.

4. If available, try a different pair of capture and detection antibodies.

5. Use a different assay format, like a competitive ELISA, to confirm that the target antigen in the diseased samples is present and structurally intact.

Best,

Malcolm Nobre

Thank you so much Malcolm! I will try these recommendations and see if they work.