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5 Factors Affecting High Background Fluorescence in Immunochromatographic Test Strips

Have you ever wondered why the test strips you make have high and unstable background values under a fluorescence immunoassay instrument, no matter how you adjust them? This article will analyze five factors that lead to high background in immunochromatographic test strips.

  1. Large microspheres with small size of NC membranes
  2. Improper control of sugar content
  3. Improper control of polymer content
  4. Improper control of salt concentration in the system
  5. Influence of buffer salt types

1. Large Microspheres with Small Pore Size of NC Membranes

This reason is easy to understand. Large-diameter microspheres slow down the chromatographic speed through small-pore membranes. Within the specified time, microspheres cannot completely chromatograph from the membrane to the absorption pad, leaving fluorescence background values on the membrane.

2. How to Understand the Cause of Sugar Content?

Many researchers like add low-concentration buffer salt + protective protein + sugar + surfactant + polymer to the conjugate pad processing solution. Although the composition is the same, the results differ due to the varying amounts used. It’s known that adding sugar to the conjugate pad processing solution can protect proteins from extreme conditions and has good hydrophilicity without participating in reactions. However, a high concentration of sugar increases the solution’s viscosity. If high concentrations of sugar are added to the conjugate pad, the viscosity of the added sample dilution increases, slowing the release of other substances in the dilution, resulting in high background as large polymers or microspheres stay on the membrane.

3. Polymer Content

High concentrations of polymers on the conjugate pad can be problematic. Some use PVP-10, some PVP K30, some PEG, and some PVA. Regardless of which is used, it’s essential to determine whether they are necessary and the appropriate molecular weight and concentration. For example, PVP K30, with a large molecular weight, has adhesive, stabilizing, and thickening effects, but high concentrations can increase solution viscosity, affecting the chromatographic effect on the membrane and resulting in high background values.

4. Salt Concentration in the Buffer System

This refers to the entire system, not just a single sample dilution or sample pad processing solution. The entire system includes the sample pad, sample dilution, conjugate pad, and microsphere reconstitution solution. Salt ion concentration affects chromatographic effects and has been verified in previous projects. This factor can serve as a direction for optimizing chromatographic effects.

5. Influence of Buffer Salt Types

It is know as a fact that different buffer salts have different effects, but some may be more suitable for certain projects than others, some may result in high/low background values. This relates to the chemical properties of the buffer salts themselves. (For more information on various buffer salts, please follow BIOEAST's social media.)


  1. To improve background values in immunochromatographic test strips, reduce sugar content (recommended 3-5%).
  2. Reduce polymer content (recommended 0.1-1%) based on specific conditions.
  3. Improve the system's salt ion concentration (around 100mMol) as needed for different projects.
  4. Changing type of the salts in buffer formulation  may provide some improvement.

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