High Background due to Non-specific binding of tetramer to CD8

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H-2K^b tetramers have been shown to exibit non-specific staining. This seems to be due to the tetramers binding to the CD8 molecule on the T-cells. To eliminate this problem a blocking anti-CD8 monoclonal antibody should be use. We highly recommend that you find the optimal concentration of both your tetramer and the anti-CD8 antibody in a cross titration experiment.

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Below are two example of a cross titration experiment with the Kb/SIINFEKL tetramer. In these experiment splenocyte from a OT-1 transgenic mouse are mixed with wildtype C57Bl/6 splenocytes at a 1:9 ratio. Titrations of the tetramer and the anti-CD8 antibody were generated and mixed together. Finally the splenocytes were incubated with the tetramer antibody cocktail.

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This experiment uses the 53-6.7 antibody (data contributed by Ms. Lily Wang).

Titration of 53-6.7 does not affect the seperation of the tetramer positive and negative CD8+ cells. However, the titration of the tetramer can yield a concentration that provided maximal seperation of the two popultations.

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This experiment uses the CD-CD8a antibody (data contributed by Ms. Lily Wang).

In this experiment it was critical to determine the optimal concentration of both the CD-CD8a antibody and the tetramer.

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