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DEPENDABLE EXCELLENCE IN VECTOR-BORNE DIAGNOSTICS

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Troubleshooting Immunofluorescence Assays

Problem: Reactions are stronger on the edges of the well in comparison to the center.

 

Possible Causes:
1. This phenomenon depends upon cell distribution within each well and the effects of the liquid droplet. As the edge of the well generally has less antigen, reactivity may appear increased due to an increase in the antibody-to-antigen ratio. This effect is amplified by the kinetic energy associated with surface tension near the edge of the serum droplet. Assays are standardized using reactivity in the central portion of the well and normal ranges are based upon this reading.

Questions / Problems

 

  1. Positive Control is negative or very weak.
  2. Antigen or substrate cells smeared or absent from wells before test.
  3. Antigen or substrate cells washed off during the procedure.
  4. Negative Control is positive.
  5. Reactions are stronger on the edges of the well in comparison to the center.