Granzyme B

 

Description

The predominant pathway used by CD8+ T cells to kill pathogen-infected cells is granule exocytosis, involving the release of Granzyme B (GB). GB is a serine protease which cleaves various caspases inside the target cell, this triggers apoptosis and death of the infected cell. GB release is a hallmark of activated cytotoxic T cells and is thus an important marker to measure in vaccine development and when studying correlates of protection against intracellular infections (Hiebert and Granville 2012).

 

Alignment

Protein alignment for marmoset, owl monkey, and squirrel monkey granzyme B:

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References

  • Hiebert, P. R. and D. J. Granville (2012). "Granzyme B in injury, inflammation, and repair." Trends Mol Med 18(12): 732-741.

 

Status

Recombinant immunogenicity-optimized marmoset Granzyme B (MAR GRZB) was expressed in HEK cells and purified using affinity chromatography. Mice were immunized with the recombinant protein, and the mice exhibiting the highest GRZB IgG titers were selected for hybridoma production. Unfortunately, the serology was weak and no positive hybridomas were identified post fusion. Thus, cross-reactivity of Mabtech’s anti-human, -bovine, -macaque, -rat, and -mouse GrzB clones against recombinant MAR GRZB were tested in ELISA. The clones identified as cross-reactive were further tested in all sandwich combinations to identify potential pairs, and then evaluated on marmoset, squirrel monkey, and owl monkey samples.

NWMIR-ELISA assay for NWM Granzyme B.
Figure 1. ELISA assay for NWM Granzyme B. Different capture/detection mAbs were used to detect GRZB in supernatants of NWM PBMC stimulated with phytohemagglutinin (PHA). A recombinant MAR GRZB was used as positive control. The figure at the bottom right shows epitope mapping for different mAb clones on human granzyme B (hGrB).

Results of the ELISA assays (Figure 1) demonstrated that several capture/detection mAb pairs were able to bind to native MAR GRZB; however, no crossreactivity with SQM or OLM GRZB was seen for these reactive pairs.

In summary, several mAbs pairs were identified that recognize native marmoset Granzyme B in an ELISA immunoassay. These mAb pairs will also be tested in a LMX format so that the selected pair can be added to a MAR-specific multiplex cocktail.