BM Chemiluminescence ELISA Substrate (POD) Troubleshooting

Product No. 11582950001


Optimizing ELISA protocols for chemiluminescence detection

POD-conjugate concentration
First use the conjugate concentration that is recommended or has been optimized for the individual assay. When the enzyme conjugate contributes to nonspecific binding, its concentration can be lowered to 1 : 10.

Nonspecific binding
Optimizing nonspecific binding is a prerequisite for highly sensitive detection. There are different nonspecific interactions that may contribute to elevated background levels (ionic interactions, hydrophobic interactions, cross-reactivity).

To reduce background:

  • Use our Blocking Reagent for ELISA.
  • Add additional components to washing, incubation, and conjugate buffer.
  • Lower the concentrations of the specific interacting components.

The following additives may be used:

  • Salt: 0.5 to 1.0 M NaCl
  • Complexing agent: 1 to 5 mM EDTA
  • Detergent: Tween 20, 0.05 to 0.1%
  • Protein: BSA, 0.1 to 1%, serum, casein, milk powder, IgG from non-crossreacting species, hydrophobic proteins

Note: In a colorimetric assay, 1% nonspecific binding accounts for approx. 0.02 OD at a maximal signal of 2.0 OD. This effect is within the range of experimental error. In a chemiluminescent assay, 1% nonspecific binding accounts for 10,000 relative light units (rlu) at a total signal of 106 rlu. In this case, two orders of magnitude in the high sensitivity range can be lost. Using Antidigoxigenin-POD, Fab-fragments results routinely in nonspecific binding in the range of 100 to 1000 rlu, varying between different ELISAs.

Low affinity antibodies
Replace low affinity antibodies by high affinity systems if possible. This can be done by conjugating a component with biotin or digoxigenin. The haptenized molecules are be bound with high affinity using Streptavidin or anti-digoxigenin antibodies.

Washing conditions
Most interactions which contribute to non-specific binding are of low/intermediate affinity and therefore reversible in character. Prolonged intervals between individual washes (we recommend at least 3 repeated washes) favor dissociation from non-specific binding sites.

Weak or no signal

  • Check instrument settings.
  • Check POD-activity of the secondary antibody.
  • Check conjugate buffer for incompatible components (e.g. NaN3, SH-reagents).
  • Check protocol (incubation times/temperatures, buffer conditions, etc.) and concentrations of primary antibody or antigen.
  • Check chemiluminescence reagent for storage conditions and biological contamination. Use freshly prepared reagent.
  • Check integrity of positive control.

High background signal

  • Prolong washing steps (number or time between steps).
  • Modulate concentrations for primary/secondary antibody.
  • Try different additives with the washing/incubation buffers to block non-specific interactions.


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